Soft, Wearable Microfluidic Systems Capable of Capture, Storage and Sensing of Biofluids

ABSTRACT

The invention provides systems for handling biofluids including the transport, capture, collection, storage, sensing, and/or evaluation of biofluids released by tissue. Systems of some aspects provide a versatile platform for characterization of a broad range of physical and/or chemical biofluid attributes in real time and over clinically relevant timeframes. Systems of some aspects provide for collection and/or analysis of biofluids from conformal, watertight tissue interfaces over time intervals allowing for quantitative temporal and/or volumetric characterization of biofluid release, such as release rates and release volumes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/625,087, filed Jun. 16, 2017, which claims the benefit of andpriority to U.S. Provisional Application Nos. 62/351,734, filed Jun. 17,2016, and 62/422,536, filed Nov. 15, 2016, each of which is herebyincorporated in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under UES S-977-02A-001UIUC subaward awarded by the U.S. Air Force. The government has certainrights in the invention.

BACKGROUND OF INVENTION

Emerging wearable sensor technologies offer attractive solutions forcontinuous, personal health/wellness assessment, forensic examination,patient monitoring and motion recognition. Recent advances in epidermalelectronics provide classes of skin-mounted sensors and associatedelectronics in physical formats that enable intimate contact with theskin for long-term, reliable health monitoring.

An important measurement mode in such devices may involve the analysisof body fluids (e.g., blood, interstitial fluid, sweat, saliva, andtear), to gain insights into various aspects of physiological health.Such function in wearable sensors, generally, and epidermal electronicsin particular, is relatively unexplored. Existing devices either usecomplex fluidic systems for sample handling or involve purelyconcentration-based measurement without sample collection and storage,or access to parameters related to quantity and rate. In addition,mechanical fixtures, straps and/or tapes that are typically required tomaintain contact of these devices with the skin do not lend themselveswell to continuous, long term monitoring without discomfort.

SUMMARY OF THE INVENTION

The invention provides systems for handling biofluids including thetransport, capture, collection, storage, sensing, and/or evaluation ofbiofluids released from by tissue. Systems of some aspects provide aversatile platform for characterization of a broad range of physicaland/or chemical biofluid attributes in real time and over clinicallyrelevant timeframes. Systems of some aspects provide for collectionand/or analysis of biofluids from conformal, watertight tissueinterfaces over time intervals allowing for quantitative temporal and/orvolumetric characterization of biofluid release, such as release ratesand release volumes. Systems of some aspects provide formulti-parametric and/or temporal profiling including tandem sensingand/or quantification of multiple analytes in biofluids as a function oftime. Systems of some aspects integrate functional substratesimplementing fluidic handling systems using biocompatible materialsproviding for time dependent fluidic capture and quantification whilemaintaining a robust tissue interface and minimizing artificial changesto the release of biofluids. Systems of some aspects, for example,integrate wireless information transfer in connection with capture,sensing and/or collection, including via near field communication andsystems capable of wireless electronic interfaces to external devices,e.g. for image capture and/or analysis. Systems of some aspects areuseful for temporal evaluation of the physical state and/or compositionof tissue, for example, for identification and/or monitoring of healthand/or the onset or progression of disease.

In some embodiments, aspects of the invention provide skin mounteddevices for temporal characterization of sweat including determinationof sweat rate, sweat volume and sweat composition as a function of time.Epidermally mounted systems of some embodiments include microfluidicnetwork geometries implemented in thin, elastic form factors andstructures providing temporally controlled fluid handling, captureand/or biochemical analytics. Epidermally mounted systems of someaspects include open architecture geometries including passages fortransporting a portion of the released sweat away from the devices so asto minimize problems with inducing irritation of the tissue and/ormaintaining a watertight interface, for example, during periods ofprofuse sweating. Epidermally mounted systems of some aspects includesystems for capture and collection of sweat for later analysis andsystems providing temporal characterization of sweat compositionincluding the concentration of electrolytes and metabolites, for examplevia colorimetric analysis, optionally for discrete analyte concentrationwindows.

In an aspect, the invention provides a device for handling a biofluidcomprising: (i) a functional substrate for mounting on a surface of theskin; and (ii) one or more sensors supported by the functionalsubstrate; wherein the functional substrate provides for microfluidictransport of at least a portion of the biofluid to the one or moresensors; and wherein the one or more sensors provide forcharacterization of at least one temporal property of the biofluid.

In an aspect, the invention provides a device for handling a biofluidcomprising: (i) a functional substrate for mounting on a surface of theskin; and (ii) a plurality of biofluid collection structures supportedby the functional substrate; wherein the functional substrate providesfor microfluidic transport of at least a portion of the biofluid to thebiofluid collection structures; and wherein each biofluid collectionstructure receives biofluid corresponding to a different time interval.

In an aspect, the invention provides a device for handling a biofluidcomprising: (i) a functional substrate for mounting on a surface of theskin; and (ii) a plurality of sensors supported by the functionalsubstrate; wherein the functional substrate provides for microfluidictransport of at least a portion of the biofluid to the plurality ofsensors; wherein the plurality of sensors include at least a firstsensor for determining a first concentration of an analyte over a firstconcentration range and a second sensor for determining a secondconcentration of the analyte over a second concentration range differentfrom the first concentration range. In an embodiment, for example, firstand second sensors each are colorimetric sensors having different colorsensitive reagents or concentrations of color sensitive regents toprovide sensitive and accurate determination of analyte concentrationsover different concentration ranges.

In an aspect, the invention provides a device for handling a biofluidcomprising: (i) a functional substrate for mounting on a surface of theskin; and (ii) one or more sensors or biofluid collection structuressupported by the functional substrate; wherein the functional substrateprovides for microfluidic transport of the biofluid including transportof a first portion of the biofluid to the one or more sensors orbiofluid collection structures and transport of a second portion of thebiofluid away from the device.

In an embodiment, a device of the invention is provided in physicalcontact with the skin of a subject. In an embodiment, a device of theinvention is provided in conformal contact with the skin of a subject.

Devices of aspects of the invention provide a versatile platformsupporting a broad range biofluid handling and manipulation applicationsincluding sample collection and in vivo diagnostics and monitoring. Inan embodiment, for example, the device is for sensing, monitoring orcharacterizing a biofluid, such as temporal characterization of one ormore physical and/or chemical properties. In an embodiment, the deviceis for capture, collecting or storing a biofluid, for example, over awell-defined sampling time interval.

Devices of aspects of the invention are useful for temporalcharacterization of biofluid release, uptake and/or composition. In anembodiment, the device is for determining a temporal property of thebiofluid, for example, characterization of physical property and/orchemical property of the biofluid as a function of time or over a knownor preselected time domain. In an embodiment, for example, the temporalproperty of the biofluid is characterized over a time domain selectedfrom the range of 10 μs to 24 hrs, optional for some applications, atime domain selected from the range of 1 ms to 24 hrs. In an embodiment,for example, the temporal property of the biofluid is a biofluid releaserate as a function of time or a biofluid release volume as a function oftime. In an embodiment, for example, the temporal property of thebiofluid is a sweat rate as a function of time or a total sweat volumeloss as a function of time. In an embodiment, for example, the temporalproperty of the biofluid is a time dependent concentration or amount ofone or more analytes in the biofluid. In an embodiment, for example, thetemporal property of the biofluid is a time dependent concentration ofone or more biomarkers in the biofluid.

In an embodiment, for example, the temporal property is amulti-parametric property including the concentrations or amounts of atleast two biomarkers as a function of time. In an embodiment, forexample, the one or more biomarkers is one or more electrolytes ormetabolites.

In an embodiment, for example, the sensors or biofluid collectionstructures are supported by a functional substrate. “Supported by afunctional substrate” may refer to a configuration wherein the sensorsor biofluid collection structures are provided directly (e.g., inphysical contact) on a surface of the functional substrate, such as anexternal surface, or provided on an intermediate structure provided on asurface of the functional substrate. “Supported by a functionalsubstrate” may also refer to a configuration wherein the sensors orbiofluid collection structures are at least partially, and optionallywholly, integrated with the functional substrate, for example, whereinat least a portion of, and optionally all, of the sensors or biofluidcollection structures comprise elements of the functional substrate. Inan embodiment, for example, the sensors or biofluid collectionstructures are integrated with the functional substrate, for example, ina configuration wherein the functional substrate provides one or morewalls or other structural elements of reservoirs, microfluidic channelsand/or chambers comprising the sensors or biofluid collectionstructures.

The disclosed devices may mobilize and access biofluids by mechanical,electrical and/or thermal mechanisms including but not limited tosurface wicking, microneedle extraction, reverse iontophoresis,capillary action and/or thermal microablasion.

In an embodiment, a device comprises at least one microneedle or anarray of microneedles for accessing interstitial fluid or blood.Microneedles may, for example, be fabricated from polymeric materials,silicon or glass using known micromachining techniques. Some methods formaking and using microneedles and microneedle arrays are described, forexample, in E. V. Mukerjee, “Microneedle array for transdermalbiological fluid extraction and in situ analysis,” Sensors and ActuatorsA, 114 (2004) 267-275. In an embodiment, a microneedle or microneedlearray may be disposed at a surface of an epidermal device, for example,at a microchannel opening.

The physical properties of the functional substrate are important inestablishing a robust interface with the tissue, such as a conformaland/or watertight interface, for example, over clinically relevanttimeframes. In an embodiment, for example, the functional substrate ismechanically matched to the skin. In an embodiment, for example, thefunctional substrate is thermally matched to the skin.

In an embodiment, a functional substrate is an elastomeric substrate. Inan embodiment, the functional substrate is substantially colorless andsubstantially transparent. In an embodiment, the functional substratehas a Young's modulus less than or equal to 100 MPa and optionally insome embodiments less than or equal to 10 MPa. In an embodiment, thefunctional substrate has a Young's modulus selected from a range of 10kPa to 10 MPa and in some embodiments selected from a range of 100 kPato 1 MPa. In an embodiment, the functional substrate has a thicknessselected from a range of 500 μm to 2 mm and in some embodiments selectedfrom a range of 500 μm to 1 mm. In some embodiments, the functionalsubstrate is selected from the group consisting of polydimethylsiloxane(PDMS), polyurethane, cellulose paper, cellulose sponge, polyurethanesponge, polyvinyl alcohol sponge, silicone sponge, polystyrene,polyimide, SU-8, wax, olefin copolymer, polymethyl methacrylate (PMMA)and polycarbonate. In some embodiments, the functional substrate has adielectric constant greater than or equal to 2.0. In some embodiments,the functional substrate has a dielectric constant selected from a rangeof 2.20 to 2.75. In some embodiments, the functional substrate haslateral dimensions or a diameter less than or equal to 700 mm². In someembodiments, the functional substrate has a permeability for thebiofluid greater than or equal to 0.2 g/h m². In some embodiments, thefunctional substrate has a coefficient of thermal expansion selectedfrom a range of 1/° C.(×10⁻⁶) to 3/° C.(×10⁻⁴). In some embodiments, thefunctional substrate has a porosity greater than or equal to 0.5. In anembodiment, for example, the functional substrate has a lateral footprint less than or equal to 1000 mm². In an embodiment, for example, thefunctional substrate has a porosity greater than or equal to 0.01,optionally for some embodiments 0.1 and optionally for some embodiments0.5. In some embodiments, devices of the invention have a porosityselected over the range of 0.1 to 0.6.

Devices of aspects of the invention include self-adhering devices anddevices that are adhered to the surface of the skin via an adhesionmaterial, such as an adhesive layer. In an embodiment, for example, thefunctional substrate is capable of adhering to the surface of the skin.In an embodiment, for example, the functional substrate adheres to thesurface of the skin with an adhesion force selected from the range of 1N to 20 N.

In an embodiment, for example, the functional substrate has one or moreinlets in fluid communication with the surface of the skin, optionallywherein the inlets are individually addressed to one or more sweatglands of the skin. In an embodiment, for example, the functionalsubstrate forms a watertight seal with the skin around the one or moreinlets.

In an embodiment, for example, the functional substrate comprises aporous material, a micro-machined material, a woven material, a meshmaterial or a fibrous material. In an embodiment, for example, thefunctional substrate comprises an adhesive layer having a plurality ofmicromachined openings. In an embodiment, for example, the functionalsubstrate comprises a microfluidic network for spatially routing atleast a portion of the biofluid. In an embodiment, for example, themicrofluidic network comprises an elastomeric material. In anembodiment, for example, the microfluidic network comprises one or moremicrochannels providing for transport of at least a portion of thebiofluid. In an embodiment, for example, the microfluidic networkcomprises a first layer embossed with a relief geometry corresponding tothe microchannels and a second top capping layer.

In an embodiment, for example, the transport of biofluid is generatedvia capillary action, natural pressure of the biofluid or a combinationof these.

In an embodiment, for example, the microfluidic network comprises aplurality of microchannels and a plurality of reservoirs, whereindifferent microchannels of the network are in fluid communication withdifferent reservoirs. In an embodiment, for example, the microchannelsand the reservoirs are connected via one or more passive valves or oneor more active valves allowing for time dependent collection, analysisor storage of the biofluid. In an embodiment, for example, themicrofluidic network further comprises one or more outlets in fluidcommunication with the microchannels for reducing backpressure in themicrofluidic network. In an embodiment, for example, the one or moreoutlets comprise openings, membranes or a combination thereof.

In an embodiment, for example, the functional substrate further compriseone or more openings providing for passage of an unsampled portion ofthe biofluid away from the device. In an embodiment, for example, theunsampled portion of the biofluid is transported away vertically orlaterally relative to the interface of the device and the skin.

In an embodiment, for example, the functional substrate comprises amaterial selected from the group consisting of polydimethylsiloxane(PDMS), polyurethane, cellulose paper, cellulose sponge, polyurethanesponge, polyvinyl alcohol sponge, silicone sponge, polystyrene,polyimide, SU-8, wax, olefin copolymer, polymethyl methacrylate (PMMA),polycarbonate or any combination.

The devices of aspects of the invention are compatible with a variety ofsensors. In an embodiment, for example, the one or more sensors comprisecolorimetric sensors. In an embodiment, for example, the one or morecolorimetric sensors comprise one or more color-responsive reagents forquantification of a volume, flow rate, composition or any combination ofthese of the biofluid. In an embodiment, for example, the one or morecolor-responsive reagents are indicator reagents that react with one ormore biomarkers in the biofluid. In an embodiment, for example, the oneor more color-responsive reagents are selected from the group consistingof CoCl₂, glucose oxidase, peroxidase, potassium iodide, lactatedehydrogenase, diaphorase, formazan dyes,2,4,6-tris(2-pyridiyl)-s-triazine (TPTZ) complexed with mercury ion oriron ion, a 2,2′-bicinchoninic acid, 1,10-phenanthroline, a universal pHindicator or any combination of these.

In an embodiment, for example, one or more color-responsive reagents areprovided in a biofluid collection structure of a microfluidic network.In an embodiment, for example, one or more color-responsive reagents areprovided in a reservoir. In an embodiment, for example, one or morecolor-responsive reagents are provided in microfluidic channel.

In an embodiment, for example, the biofluid collection structure of themicrofluidic network is at least partially optically transparent in thevisible or infrared region of the electromagnetic spectrum. In anembodiment, for example, the biofluid collection structure having thecolor-responsive reagents is characterized by a volume selected over therange of 1000 μm³-1000 mm³. In an embodiment, for example, one or morecolor-responsive reagents are immobilized within the biofluid collectionstructure. In an embodiment, for example, one or more color-responsivereagents are immobilized within or on one or more walls of the biofluidcollection structure. In an embodiment, for example, one or morecolor-responsive reagents are immobilized within a hydrogel providedwithin the biofluid collection structure.

In an embodiment, for example, the color-responsive reagents areprovided along the length of the biofluid collection structurecomprising a microfluidic channel, wherein the volume of the biofluid inthe microchannel is sensed as the biofluid fills the microchannel. In anembodiment, for example, a leading edge of the volume of biofluid in themicrochannel is sensed (e.g., optically, visually, mechanically,electrochemically, chemically, etc.) as a function of time. In anembodiment, for example, the leading edge of the volume of the biofluidin the microchannel is sensed optically. In an embodiment, for example,the microchannel is a serpentine microchannel, thereby providingimproved sensitivity over a larger operating range without undulyincreasing substrated footprint area.

In an embodiment, for example, the color-responsive reagent is providedin the biofluid collection structure, and wherein the concentration ofthe one or more biomarkers in the biofluid are sensed as the biofluid isprovided to the biofluid collection structure. In an embodiment, forexample, the concentrations of the one or more biomarkers in thebiofluid are sensed optically, optionally visually or with a camera.

In an embodiment, for example, the one or more biofluid collectionreservoirs are one or more conduits and/or reservoirs, or are one ormore reservoirs. In an embodiment, for example, the one or more biofluidcollection reservoirs are one or more chambers. In an embodiment, forexample, the one or more biofluid collection reservoirs are one or moremicrofluidic channels. In an embodiment, for example, the microfluidicchannels are characterized by a length selected from a range of 1 mm to50 cm. In an embodiment, for example, the microfluidic channels arecharacterized a cross sectional area selected from a range of 100 μm² to10 mm².

In an embodiment, for example, the device is read-out passively, forexample, by a passive optical, mechanical or electronic signal. In anembodiment, for example, the device is read-out actively, for example,wherein the device actively generates an NFC signal.

In an embodiment, for example, wherein the device is for collecting,storing or analyzing the biofluid. In an embodiment, for example, thebiofluid is sweat, blood or interstitial fluid from a subject. In anembodiment, for example, the biofluid is selected from the groupconsisting of sweat, tears, saliva, gingival crevicular fluid,interstitial fluid, blood and combinations thereof.

In an embodiment, for example, wherein the device is for determining theconcentration in one or more biomarkers in the biofluid. In anembodiment, for example, the one or more biomarkers in the biofluid areelectrolytes or metabolites.

In an embodiment, for example, the device further comprises an actuator.In an embodiment, for example, the actuator generates electromagneticradiation, acoustic energy, an electric field, a magnetic field, heat, aRF signal, a voltage, a chemical change or a biological change. In anembodiment, for example, the actuator comprises a heater, a reservoircontaining a chemical agent capable of causing a chemical change or abiological change, a source of electromagnetic radiation, a source of anelectric field, a source of RF energy or a source of acoustic energy. Inan embodiment, for example, the device further comprises a transmitter,receiver or transceiver. In an embodiment, for example, the devicefurther comprises at least one coil. In an embodiment, for example, theat least one coil is a near-field communication coil. In an embodiment,for example, the at least one coil is an inductive coil. In anembodiment, for example, the at least one coil comprises a serpentinetrace.

In an embodiment, for example, the device has an average Young's modulusand a thickness within a factor of 2 of a modulus and a thickness of anepidermal layer of the skin of a subject. In an embodiment, for example,the device has an average Young's modulus less than or equal to 500 kPa.In an embodiment, for example, the device has an average Young's modulusselected from a range of 0.5 kPa to 100 kPa. In an embodiment, forexample, the device has a net bending stiffness less than or equal to 1nN m. In an embodiment, for example, the device has a net bendingstiffness selected from a range of 0.1 to 1 nN m. In an embodiment, forexample, the device has a 2D footprint (e.g., area of the skininterface) selected from a range of 300 mm² to 2000 cm².

In an aspect, provided herein are methods of analyzing a biofluid; themethods comprising: (i) providing a device for handling a biofluid; thedevice comprising: (1) a functional substrate for mounting on a surfaceof the skin; and (2) one or more sensors supported by the functionalsubstrate; wherein the functional substrate provides for microfluidictransport of at least a portion of the biofluid to the one or moresensors; and wherein the one or more sensors provide forcharacterization of at least one temporal property of the biofluid; (ii)contacting the functional substrate of the device with a surface of theskin of a subject; and (iii) analyzing the biofluid from the surface ofthe skin of the subject.

In an aspect, provided herein are methods of collecting a biofluid; themethods comprising: (i) providing a device for handling a biofluid; thedevice comprising: (1) a functional substrate for mounting on a surfaceof the skin; and (2) a plurality of biofluid collection structuressupported by the functional substrate; wherein the functional substrateprovides for microfluidic transport of at least a portion of thebiofluid to the biofluid collection structures; and wherein eachbiofluid collection structure receives biofluid corresponding to adifferent time interval; (ii) contacting the functional substrate of thedevice with a surface of the skin of a subject; and (iii) collecting thebiofluid from the surface of the skin of the subject.

In an aspect, provided herein are methods of analyzing a biofluid; themethods comprising: (i) providing a device for handling a biofluid; thedevice comprising: (1) a functional substrate for mounting on a surfaceof skin; and (2) a plurality of sensors supported by the functionalsubstrate; wherein the functional substrate provides for microfluidictransport of at least a portion of the biofluid to the plurality ofsensors; wherein the plurality of sensors include at least a firstsensor for determining a first concentration of an analyte over a firstconcentration range and a second sensor for determining a secondconcentration of the analyte over a second concentration range differentfrom the first concentration range; (ii) contacting the functionalsubstrate of the device with a surface of the skin of a subject; and(iii) analyzing the biofluid from the surface of the skin of thesubject.

In an aspect, provided herein are methods of sampling a biofluid; themethods comprising: (i) providing a device for handling a biofluid; thedevice comprising: (1) a functional substrate for mounting on a surfaceof the skin; and (2) one or more sensors or biofluid collectionstructures supported by the functional substrate; wherein the functionalsubstrate provides for microfluidic transport of the biofluid includingtransport of a first portion of the biofluid to the one or more sensorsor biofluid collection structures and transport of a second portion ofthe biofluid away from the device; (2) contacting the functionalsubstrate of the device with a surface of the skin of a subject; and (3)sampling the biofluid from the surface of the skin of the subject.

As will be understood by one of skill in the art, any of the devices,device components and device features described herein can be used inthe present methods. In an embodiment, for example, the biofluid issweat in a method of the invention. In an embodiment, for example, themethod determines a temporal characteristic of the sweat. In anembodiment, for example, the method determines a compositionalcharacteristic of the sweat.

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematics, optical images, and theoretical stress modeling ofan epidermal microfluidic biosensor integrated with flexible electronicsfor sweat monitoring. (A) Schematic illustration of an epidermalmicrofluidic sweat monitoring device and an enlarged image of theintegrated near-field communication (NFC) system (panel A2). (B)Illustration of the top, middle, and back sides of a device. Thereference color (white and black) markers are on the top side, alongwith the NFC electronics. The microfluidic channels with colorimetricassay reagents (water, lactate, chloride, glucose, and pH) are in themiddle. The bottom side consists of a uniform layer of adhesive bondedto the bottom surfaces of the PDMS-enclosed microchannels with openingsthat define sweat access and openings that connect to these channels.(C) Cross-sectional diagrams of the cuts defined by the dashed lines (a)and (b) shown in the top side illustration in (B). (D) Optical image ofa fabricated epidermal microfluidic sensor (E) Calculated finite elementanalysis (FEA) results of stress distribution on the devices on phantomskin (PDMS) and respective optical images under various mechanicaldistortions: stretching at 30% strain, bending with 5 cm radius, andtwisting with skin.

FIG. 2. Analysis of key design features and demonstration of epidermalmicrofluidic patches. (A) Sketch of the channel geometry for numericalcalculation. The blue and red dashed boxes highlight the dimensions ofthe serpentine and outlet channels, respectively. (B) Experimentallydetermined water vapor loss from a microfluidic channel as a function ofwidth (w) and length (L) of the outlet channel with a fixed height of300 μm. Inner pressure variation as a function of the outlet channelwidth was also determined from the model (red line). The orange shadinghighlights the optimal channel geometry. Data are presented as theaverage value, and error bars represent standard deviation (n=3). (C)Model prediction of the change in volume of the serpentine channel as afunction of aspect ratio (ratio of width ‘a’ to height ‘h’ of theserpentine channel in (A), blue dashed box) under various pressures(ΔP=100, 200 and 400 Pa). ΔP represents pressure difference between theinside and outside of the serpentine channel. Dotted vertical lines showtwo representative aspect ratios (10:3 and 5:1). (D) Picture of afabricated epidermal microfluidic structure corresponding to thetheoretical results and cross-sectional scanning electron microscope(SEM) images of the outlet (red dashed box) and serpentine (blue dashedbox) channels. (E) Experimental set-up of artificial sweat pore system.(F) SEM images of the polyimide (PI) membrane mimicking human sweatglands. (G) Demonstration of hydrodynamic fluid flow through themicrofluidic device using the artificial sweat pore system at the rateof 5.5 μL/h.

FIG. 3. Quantitative colorimetric analysis of markers in sweat. (A)Colorimetric detection reservoirs that enable determination of (B) totalwater (sweat) loss and concentrations of (C) lactate, (D) glucose, (E)creatinine, (F) pH, and (G) chloride ions in sweat. (B-G) Correspondingquantitative analysis conducted by (i) UV-vis spectroscopy and (ii)optical images as a function of analyte concentrations. The presentedcolor for (i) each spectrum corresponds to (ii) the color exhibited atthe detection reservoir in the device. The insets in the spectra providecalibration curves for each of the analytes. The inset in (E) shows theresponse over a reduced range of concentrations.

FIG. 4. Near field communication interface to a smartphone and imageprocessing. (A) Pictures demonstrating near field communication betweensweat monitoring device and a smart phone to launch software for imagecapture and analysis. (B) Images of the epidermal microfluidic biosensor(left) before and (right) after injecting artificial sweat. (C) Locationtracking of sweat accumulation with polar coordinates and theirrelationship to total captured volume of sweat (inset). (D) Standardcalibration curves between normalized % RGB value and concentration ofmarkers for quantitative analysis (n=3, error bars represent thestandard deviation). Each vertical colored bar represents the markerconcentration determined from the corresponding reservoirs in the rightimage of (B) as an example.

FIG. 5. Human trials of sweat monitoring devices in a temperature andhumidity controlled room (35° C. at 50% relative humidity). (A) Adhesivelayers utilized for human studies in a controlled setting. Brown colorcorresponds to the adhesive layer on the backside of the device withsmall and large harvesting areas (inlets). Absorbing pads served as areference control. (B) An illustration indicating locations of sweatpatches on the subjects (volar forearm and lower back). (C) Images oftwo different types of sweat patches (small and large harvesting areas)applied to the lower back and volar forearm according to study periods.(D) Difference of sweat rate according to body areas (lower back andvolar forearm). Bars represent mean of n=8, error bars SD. *p<0.05,two-tailed t-test. (E) Correlation of sweat rate between the sweatpatches and the reference-absorbing pads (n=7). (F) Markerconcentrations in sweat obtained by image processing of data from thedevice (un-shaded) versus lab-based analysis of sweat collected fromabsorbing pads (shaded) (n=7). *p<0.05, two-tailed t-test.

FIG. 6. Analysis of sweat monitoring devices on bikers in anuncontrolled environment. (A) An illustration indicating locations ofsweat patches on the cycling subjects (volar forearm and lower back).(B) Histogram showing age distribution of cycling subjects. (C) Trendsof temperature and humidity change during the race. (D) Elevationprofile of the course. (E) Sweat patches on the volar forearms of studysubjects, imaged after ˜84 km of cycling (i.e., middle point of totalrace). (The purple ink in the lower part of the image on the right isfrom a marking formed on the skin using a pen, prior to application ofthe device.) (F) Sweat patches on the volar forearms of young femalesubject

FIG. 7. Fabrication procedures of the epidermal microfluidic deviceusing soft lithography.

FIG. 8. Determination of adhesion forces and conformal adhesion betweendevice and skin. (A) Experimental set-up for 90° peel adhesion propertytesting (standard ISO 29862:2007) using a force gauge (Mark-10,Copiague, N.Y.). Images of (B) holding a device adhered on the skin withforce gauge and (C) peeling devices at an angle of 90°. (D) Forcemeasurement while displacing the device at the rate of 300 mm/minindicating the gray region where peeling was occurred. Determinedaverage peeling force was 5.7 N. Sweat patches conformably adhere undervarious conditions, such as applying (E) no strain, (F) stretching, (G)compressing, and (H) twisting distortions as well as laminating on (I)sweaty and (J) hairy skin.

FIG. 9. Observations of sweat at the interface between an adhesive layerand the skin. (A) A picture of a piece of medical adhesive on the skinand (B-C) images of the sweat trapped under the adhesive duringexercise. (D-F) Images collected at various times during a resting stateimmediately after the exercise. The sweat appears in isolated pockets,and gradually reabsorbs into the skin, consistent with negligiblelateral flow. The reabsorption rate evaluated from such experiments was˜12 mg/cm² h, while the moisture vapor transmission rate (MVTR) of theacrylic adhesive is 2.08 mg/cm² h.

FIG. 10. Normal (A) and shear (B) stress distribution at device/skininterface under 30% stretch plotted on deformed skin.

FIG. 11. Mechanical modeling results for NFC electronics. (A)Stretching, (B) bending, and (C) twisting deformations.

FIG. 12. Color balancing performed by internal calibration makers (blackcrosses and white circle) under various light conditions (A-F) andchanges in numeric RGB representation obtained by respective images (G)before and (H) after white balance. Images before (left) and after(right) color calibration performed under various light conditions,including (A) sun light, (B) shadow, (C) incandescent, (D) fluorescence,(E) LED, and (F) halogen bulb light.

FIG. 13. Image processing for position calibration. (A) Original imageand magnified images (B) before and (C) after position calibration.

FIG. 14. Calculation of the inner pressure as a function of the outletchannel length.

FIG. 15. Strategies and optimization of the orbicular channel design.(A) Illustration of four different types of channel designs. Red brokenlines represent a reference circle (r=15 mm) that corresponds to theouter edge of the sweat patch. Blue broken lines show neural circles ofvarious designs of orbicular channels. (B) A comparative table showingquantitative values for each channel design.

FIG. 16. Schematic illustration of the artificial sweat pore system.

FIG. 17. Hydrodynamic test to verify the influence of the hydrogelmatrix on channel volume. (A) Picture of the experimental set-up. Asyringe pump introduced 15 μL of water with red dye into themicrofluidic device at the rate of 120 μL/hour. Optical images ofdevices consisting of (B) blank channel and the channels coated (C)without and (D) with CoCl2 embedded in the thin (25 μm) hydrogel matrix.(E) Images of devices with and without hydrogel coatings on theserpentine channel at the certain period incubation time. (E) Angularposition of the change in color of the serpentine channel (black) andrespective reading of the harvested volume of artificial sweat in thedevice (blue) as a function of incubation time. (F) Plot of changes onthe angle of the filling vs Incubation time (h).

FIG. 18. Assessment of the angular position of the liquid front inpartially filled serpentine channels in devices with different hydrogelconcentrations and segmented hydrogel patterns. (A) Images of devices atvarious times after partially filling the serpentine channels. (B)Changes of angular position of the leading edge and (C) respectivereading of harvested volume as a function of incubation time for thesevarious cases.

FIG. 19. Quantitative colorimetric analysis of glucose detection at lowconcentrations. (A) Standard calibration curve of normalized % RGB as afunction of glucose concentration and (B) corresponding optical imagesof color in the detection reservoir.

FIG. 20. Colorimetric analysis of device response as a function of timeafter introduction of artificial sweat. (A) Optical images of a devicemounted on a glass slide on a white background collected hourly during a6 hour period. (B) Corresponding % RGB information collected from thefour biomarker detection reservoirs. (C) Relative changes ofconcentration normalized by the initial values via image analysis.

FIG. 21. Cross sectional sketch of the microfluidic channel and outletchannel geometry used for analytical analysis of backpressure. (A) Theair inside the microfluidic channel obeys the ideal gas law, and (B) therelation between pressure drop ΔP and air escape rate is determined fromfluid dynamics analysis of the outlet channel. (C) Cross sections viewof the outlet channel.

FIG. 22. Various device configurations. The sweat patch supportscapabilities in detecting four difference biomarkers, includingchloride, pH, lactate, and (A) glucose or (B) creatinine, where thelatter two regions occur in the bottom right corners of the devices(indicated with blue dotted line). The near-field communicationelectronics can facilitate (C) image capture and (D) temperature sensingby use of M24LR04E (ST Microelectronics) and SL13A (AMS AG) chips,respectively (indicated with red dotted line).

FIG. 23. Multivariate statistical analysis for correlations in biomarkerconcentrations between patch (p) and lab (l) analysis. (A) Pearsoncorrelation map. (B) Spearman rank-order statistic.

FIG. 24. (a) Schematic illustrations and optical images (inset) of thin,soft (i.e. epidermal) skin-mounted microfluidic systems forchrono-sampling of sweat. (b) Exploded schematic illustration of adevice. (c) Top view illustration of microfluidic channels filled withdyed water. (d) Optical images of an in vitro test of sequentialsampling of dyed water introduced into a device at constant pressure.

FIG. 25. (a) Detailed schematic illustration of a unit cell of a device,showing a collection chamber, evaluation chamber, sampling outlet andthree capillary bursting valves (CBV) and SEM images of CBVs. (b) Sketchof capillary bursting valves with channel width and diverging angleindicated. (c) Measurements of the water contact angle of a PDMS surfaceas a function of time after treatment with an oxygen plasma. (d)Experimental results (bars) and theoretical values (square box) ofcapillary bursting pressures of CBV #1, #2 and #3 of a typical device.(e) Optical images and schematic illustrations of the operation ofcapillary bursting valves for chrono-sampling. i) before entering thecollection chamber ii) filling the collection chamber iii) flowing tonext chamber iv) after centrifugation.

FIG. 26. Optical images of (a) in vitro test of chrono-sampling of dyedwater introduced into 12 collection chambers, (b) in vitro test ofchrono-sampling of different color dyes in water (red, green and blue)with enlarged image of the interface between adjacent colors and afterseparation by centrifugation, (c) chrono-sampling device filled withdyed water and attached to the skin under various mechanicaldistortions: stretching, compressing, twisting and undergoing rapidmotion, (d) removing a device filled with dyed water and attached to theskin.

FIG. 27. Optical images of (a) devices with various sizes andconfigurations, each filled with dyed water. The sizes range from 1 cmto 4 cm in diameter, with numbers of chambers from 3 to 24, (b) devicesat various mounting positions across the body; behind the ear, chest,back, forearm and thigh, (c) device with sweat guiding channels on itsbottom side, as a mechanism for release of sweat from regions under thedevice but away from the inlet, (d) device with additional set ofchannels and chambers in the center, (e) device immediately aftersampling of dyed water (0 h) and after 3 days of storage while heldusing a clamp designed to prevent evaporation through the outletchannels.

FIG. 28. In situ perspiration analysis from various body positionsduring running exercise and thermal exposure in a sauna. Chrono-samplingof sweat generated on the forearm during a running exercise underconstant load (a) in thermal exposure at 56° C. (b). Cobalt (II)chloride dissolved in pHEMA and loaded into the devices aids invisualization of the filling process. Chemical analysis of lactate,sodium and potassium in the sweat extracted from a chrono-samplingdevice mounted on the forearm during a running exercise (c), fromforearm (d), thigh (e), back (f) and chest (g) in thermal exposure. (h)Regional variations of biomarker concentrations in sweat (lactate,sodium and potassium) collected from different body positions; chest,thigh, forearm and back.

FIG. 29. Fabrication procedures of the epidermal microfluidic device.

FIG. 30. Chemical contamination test in chrono-sampling device. (a)hydrostatic pressure generator fills the PDMS long channel. (b)quantitative chemical analysis of calcium, potassium, sodium andchloride in artificial sweat from PDMS device and control. (c)qualitative chemical analysis of glucose, urea in artificial sweat fromPDMS device and control.

FIG. 31. Rounded edge of the capillary bursting valve (CBV). (a) actualdescription of the valve with round edges. The radius of the roundededge of CBV #2 (b) and CBV #3 (c).

FIG. 32. a) chrono-sampling device with thorough hole in the device orleak out channel for guiding the sweat not for analysis b)chrono-sampling device with cutout in the middle. c) various device withdifferent volume of chamber from 2.3 to 6.1

FIG. 33. Mechanical clamp for blocking evaporation (a) evaporation rateof chrono-sampling device filled with dye in water. (b) evaporation testof chrono-sampling device filled with dye in water. (c) blocking regionin the device. (d) exploded schematic illustration of mechanical clampand PDMS device. (e) cross-section illustration of working principle ofclamp (f) the device filled with due in water before and after clamping.The wall in the clamp block the channel in the device.

FIG. 34. Sweat rate measurement test. Top panel shows an exemplarydevice for calculating the sweat rate from conventional method andchrono-sampling device. Bottom panel illustrates enhanced sweat in theskin around the area blocked by adhesive. The sweat rate can be measuredby the chrono-sampling device and conventional method

FIG. 35. Overall view of the device illustration and optical image. (A)The exploded structure of device. (B) Assembled device, and logicaloperation. (C) The expansion of unit logical channel, and chemical, andmechanical functions. Arrows indicate the sequence order of sweat flow.

FIG. 36. Quantitative colorimetric assays. (A) The effect of Tween 80 onthe stabilization of color development in TPTZ competition reaction forchloride assay. (B) Color index of blue color development under whitelight source. (C) Color index of blue color development under yellowlight source. (D) Comparison of (B) and (C) after normalization of theRGB index using eq. 1. (E) The calibration curve of RGB (up), andnormalization (middle) and the optical color samples (bottom) forquantitative analysis of chloride. (F) Plot of intensity vs. chlorideconcentration (mM).

FIG. 37. Mechanical strategies to support accurate colorimetric assay.(A) The design of selective SAP valve, red arrows are the sweat flow.(B) The effect of selective SAP valve, red arrow is the sweat flow.

FIG. 38. Chrono-sampling of sweat. (A) Schematic experiment design forthe direction selecting valve in microfluidics. (B) The effect ofhydrophobic treatment for designated flow. (C) Chrono-sampling manner ofthe sweat flow in the microfluidics. (D) The operation steps in the timesequence. (E) Chrono-sampling of designated sweat flow using selectiveSAP valve and hydrophobic valve. (F) Optical image of thechrono-sampling in time course. (G) Effect of pumping pressure on thesampling time. (H) Effect of inlet size on the sampling time.

FIG. 39. Practical human study for detection of biomarkers in sweat insitu. (A) Picture of controlled room. (B) Illustration and pictures forsweat patch location on body. (C) The results of sweat assay in situ:The change of l-lactate (C1) (D) The result for subject 1.

FIG. 40. An illustration of a microfluidic network having a first layerembossed on a substrate surface to provide relief geometry to definemicrochannels and a second layer that is a top capping layer.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

“Functional substrate” refers to a substrate component for a devicehaving at least one function or purpose other than providing mechanicalsupport for a component(s) disposed on or within the substrate. In anembodiment, a functional substrate has at least one skin-relatedfunction or purpose. In an embodiment, a functional substrate of thepresent devices and methods exhibits a microfluidic functionality, suchas providing transport of a bodily fluid through or within thesubstrate, for example via spontaneous capillary action or via an activeactuation modality (e.g. pump, etc.). In an embodiment, a functionalsubstrate has a mechanical functionality, for example, providingphysical and mechanical properties for establishing conformal contact atthe interface with a tissue, such as skin. In an embodiment, afunctional substrate has a thermal functionality, for example, providinga thermal loading or mass small enough so as to avoid interference withmeasurement and/or characterization of a physiological parameter, suchas the composition and amount of a biological fluid. In an embodiment, afunctional substrate of the present devices and method is biocompatibleand/or bioinert. In an embodiment, a functional substrate may facilitatemechanical, thermal, chemical and/or electrical matching of thefunctional substrate and the skin of a subject such that the mechanical,thermal, chemical and/or electrical properties of the functionalsubstrate and the skin are within 20%, or 15%, or 10%, or 5% of oneanother.

In some embodiments, a functional substrate that is mechanically matchedto a tissue, such as skin, provides a conformable interface, forexample, useful for establishing conformal contact with the surface ofthe tissue. Devices and methods of certain embodiments incorporatemechanically functional substrates comprising soft materials, forexample exhibiting flexibility and/or stretchability, such as polymericand/or elastomeric materials. In an embodiment, a mechanically matchedsubstrate has a modulus less than or equal to 100 MPa, and optionallyfor some embodiments less than or equal to 10 MPa, and optionally forsome embodiments, less than or equal to 1 MPa. In an embodiment, amechanically matched substrate has a thickness less than or equal to 0.5mm, and optionally for some embodiments, less than or equal to 1 cm, andoptionally for some embodiments, less than or equal to 3 mm. In anembodiment, a mechanically matched substrate has a bending stiffnessless than or equal to 1 nN m, optionally less than or equal to 0.5 nN m.

In some embodiments, a mechanically matched functional substrate ischaracterized by one or more mechanical properties and/or physicalproperties that are within a specified factor of the same parameter foran epidermal layer of the skin, such as a factor of 10 or a factor of 2.In an embodiment, for example, a functional substrate has a Young'sModulus or thickness that is within a factor of 20, or optionally forsome applications within a factor of 10, or optionally for someapplications within a factor of 2, of a tissue, such as an epidermallayer of the skin, at the interface with a device of the presentinvention. In an embodiment, a mechanically matched functional substratemay have a mass or modulus that is equal to or lower than that of skin.

In some embodiments, a functional substrate that is thermally matched toskin has a thermal mass small enough that deployment of the device doesnot result in a thermal load on the tissue, such as skin, or smallenough so as not to impact measurement and/or characterization of aphysiological parameter, such as a characteristic of a biological fluid(e.g. composition, rate of release, etc.). In some embodiments, forexample, a functional substrate that is thermally matched to skin has athermal mass low enough such that deployment on skin results in anincrease in temperature of less than or equal to 2 degrees Celsius, andoptionally for some applications less than or equal to 1 degree Celsius,and optionally for some applications less than or equal to 0.5 degreeCelsius, and optionally for some applications less than or equal to 0.1degree Celsius. In some embodiments, for example, a functional substratethat is thermally matched to skin has a thermal mass low enough that itdoes not significantly disrupt water loss from the skin, such asavoiding a change in water loss by a factor of 1.2 or greater.Therefore, the device does not substantially induce sweating orsignificantly disrupt transdermal water loss from the skin.

In an embodiment, the functional substrate may be at least partiallyhydrophilic and/or at least partially hydrophobic.

In an embodiment, the functional substrate may have a modulus less thanor equal to 100 MPa, or less than or equal to 50 MPa, or less than orequal to 10 MPa, or less than or equal to 100 kPa, or less than or equalto 80 kPa, or less than or equal to 50 kPa. Further, in someembodiments, the device may have a thickness less than or equal to 5 mm,or less than or equal to 2 mm, or less than or equal to 100 μm, or lessthan or equal to 50 μm, and a net bending stiffness less than or equalto 1 nN m, or less than or equal to 0.5 nN m, or less than or equal to0.2 nN m. For example, the device may have a net bending stiffnessselected from a range of 0.1 to 1 nN m, or 0.2 to 0.8 nN m, or 0.3 to0.7 nN m, or 0.4 to 0.6 nN m.

A “component” is used broadly to refer to an individual part of adevice.

“Sensing” refers to detecting the presence, absence, amount, magnitudeor intensity of a physical and/or chemical property. Useful devicecomponents for sensing include, but are not limited to electrodeelements, chemical or biological sensor elements, pH sensors,temperature sensors, strain sensors, mechanical sensors, positionsensors, optical sensors and capacitive sensors.

“Actuating” refers to stimulating, controlling, or otherwise affecting astructure, material or device component. Useful device components foractuating include, but are not limited to, electrode elements,electromagnetic radiation emitting elements, light emitting diodes,lasers, magnetic elements, acoustic elements, piezoelectric elements,chemical elements, biological elements, and heating elements. In thecontext of communications, actuating may refer to a NFC chip useful inproviding communication capability to and/or from the electronicsportion of any of the devices provided herein.

The terms “directly and indirectly” describe the actions or physicalpositions of one component relative to another component. For example, acomponent that “directly” acts upon or touches another component does sowithout intervention from an intermediary. Contrarily, a component that“indirectly” acts upon or touches another component does so through anintermediary (e.g., a third component).

“Encapsulate” refers to the orientation of one structure such that it isat least partially, and in some cases completely, surrounded by, orembedded in, one or more other structures, such as a substrate, adhesivelayer or encapsulating layer. “Partially encapsulated” refers to theorientation of one structure such that it is partially surrounded by oneor more other structures, for example, wherein 30%, or optionally 50%,or optionally 90% of the external surface of the structure is surroundedby one or more structures. “Completely encapsulated” refers to theorientation of one structure such that it is completely surrounded byone or more other structures.

“Dielectric” refers to a non-conducting or insulating material.

“Polymer” refers to a macromolecule composed of repeating structuralunits connected by covalent chemical bonds or the polymerization productof one or more monomers, often characterized by a high molecular weight.The term polymer includes homopolymers, or polymers consistingessentially of a single repeating monomer subunit. The term polymer alsoincludes copolymers, or polymers consisting essentially of two or moremonomer subunits, such as random, block, alternating, segmented,grafted, tapered and other copolymers. Useful polymers include organicpolymers or inorganic polymers that may be in amorphous, semi-amorphous,crystalline or partially crystalline states. Crosslinked polymers havinglinked monomer chains are useful for some applications. Polymers useablein the methods, devices and components disclosed include, but are notlimited to, plastics, elastomers, thermoplastic elastomers,elastoplastics, thermoplastics and acrylates. Exemplary polymersinclude, but are not limited to, acetal polymers, biodegradablepolymers, cellulosic polymers, fluoropolymers, nylons, polyacrylonitrilepolymers, polyamide-imide polymers, polyimides, polyarylates,polybenzimidazole, polybutylene, polycarbonate, polyesters,polyetherimide, polyethylene, polyethylene copolymers and modifiedpolyethylenes, polyketones, poly(methyl methacrylate),polymethylpentene, polyphenylene oxides and polyphenylene sulfides,polyphthalamide, polypropylene, polyurethanes, styrenic resins,sulfone-based resins, vinyl-based resins, rubber (including naturalrubber, styrene-butadiene, polybutadiene, neoprene, ethylene-propylene,butyl, nitrile, silicones), acrylic, nylon, polycarbonate, polyester,polyethylene, polypropylene, polystyrene, polyvinyl chloride,polyolefin, polydimethylsiloxane, polysodiumacrylate or any combinationsof these.

“Elastomer” refers to a polymeric material which can be stretched ordeformed and returned to its original shape without substantialpermanent deformation. Elastomers commonly undergo substantially elasticdeformations. Useful elastomers include those comprising polymers,copolymers, composite materials or mixtures of polymers and copolymers.Elastomeric layer refers to a layer comprising at least one elastomer.Elastomeric layers may also include dopants and other non-elastomericmaterials. Useful elastomers include, but are not limited to,thermoplastic elastomers, styrenic materials, olefinic materials,polyolefin, polyurethane thermoplastic elastomers, polyamides, syntheticrubbers, PDMS, polybutadiene, polyisobutylene,poly(styrene-butadiene-styrene), polyurethanes, polychloroprene andsilicones. Exemplary elastomers include, but are not limited to siliconcontaining polymers such as polysiloxanes including poly(dimethylsiloxane) (i.e. PDMS and h-PDMS), poly(methyl siloxane), partiallyalkylated poly(methyl siloxane), poly(alkyl methyl siloxane) andpoly(phenyl methyl siloxane), silicon modified elastomers, thermoplasticelastomers, styrenic materials, olefinic materials, polyolefin,polyurethane thermoplastic elastomers, polyamides, synthetic rubbers,polyisobutylene, poly(styrene-butadiene-styrene), polyurethanes,polychloroprene and silicones. In an embodiment, a polymer is anelastomer.

“Conformable” refers to a device, material or substrate which has abending stiffness that is sufficiently low to allow the device, materialor substrate to adopt a useful contour profile, for example a contourprofile allowing for conformal contact with a surface having surfacefeatures, e.g. relief or recessed features. In certain embodiments, adesired contour profile is that of skin.

“Conformal contact” refers to contact established between a device and areceiving surface. In one aspect, conformal contact involves amacroscopic adaptation of one or more surfaces (e.g., contact surfaces)of a device to the overall shape of a surface. In another aspect,conformal contact involves a microscopic adaptation of one or moresurfaces (e.g., contact surfaces) of a device to a surface resulting inan intimate contact substantially free of voids. In an embodiment,conformal contact involves adaptation of a contact surface(s) of thedevice to a receiving surface(s) such that intimate contact is achieved,for example, wherein less than 20% of the surface area of a contactsurface of the device does not physically contact the receiving surface,or optionally less than 10% of a contact surface of the device does notphysically contact the receiving surface, or optionally less than 5% ofa contact surface of the device does not physically contact thereceiving surface.

“Young's modulus” is a mechanical property of a material, device orlayer which refers to the ratio of stress to strain for a givensubstance. Young's modulus may be provided by the expression:

$\begin{matrix}{{E = {\frac{({stress})}{({strain})} = {\left( \frac{L_{0}}{\Delta L} \right)\left( \frac{F}{A} \right)}}},} & (I)\end{matrix}$

where E is Young's modulus, L₀ is the equilibrium length, ΔL is thelength change under the applied stress, F is the force applied, and A isthe area over which the force is applied. Young's modulus may also beexpressed in terms of Lame constants via the equation:

$\begin{matrix}{{E = \frac{\mu \left( {{3\lambda} + {2\mu}} \right)}{\lambda + \mu}},} & ({II})\end{matrix}$

where λ and μ are Lame constants. High Young's modulus (or “highmodulus”) and low Young's modulus (or “low modulus”) are relativedescriptors of the magnitude of Young's modulus in a given material,layer or device. In some embodiments, a high Young's modulus is largerthan a low Young's modulus, preferably about 10 times larger for someapplications, more preferably about 100 times larger for otherapplications, and even more preferably about 1000 times larger for yetother applications. In an embodiment, a low modulus layer has a Young'smodulus less than 100 MPa, optionally less than 10 MPa, and optionally aYoung's modulus selected from the range of 0.1 MPa to 50 MPa. In anembodiment, a high modulus layer has a Young's modulus greater than 100MPa, optionally greater than 10 GPa, and optionally a Young's modulusselected from the range of 1 GPa to 100 GPa. In an embodiment, a deviceof the invention has one or more components having a low Young'smodulus. In an embodiment, a device of the invention has an overall lowYoung's modulus.

“Low modulus” refers to materials having a Young's modulus less than orequal to 10 MPa, less than or equal to 5 MPa or less than or equal to 1MPa. In some embodiments, the functional substrate is a low modulusmaterial, such as a low modulus elastomer.

“Bending stiffness” is a mechanical property of a material, device orlayer describing the resistance of the material, device or layer to anapplied bending moment. Generally, bending stiffness is defined as theproduct of the modulus and area moment of inertia of the material,device or layer. A material having an inhomogeneous bending stiffnessmay optionally be described in terms of a “bulk” or “average” bendingstiffness for the entire layer of material.

The invention can be further understood by the following non-limitingexamples.

EXAMPLE 1 Soft, Wearable Microfluidic Systems Capable of Capture,Storage, and Colorimetric Sensing of Sweat

Capabilities in health monitoring via capture and quantitative chemicalanalysis of sweat could complement, or potentially obviate the need for,approaches based on sporadic assessment of blood samples. Establishedsweat monitoring technologies use simple fabric swatches and are limitedto basic analysis in controlled laboratory or hospital settings. Here,we present a collection of materials and device designs for soft,flexible and stretchable microfluidic systems, including embodimentsthat integrate wireless communication electronics, which can intimatelyand robustly bond to the surface of skin without chemical or mechanicalirritation. This integration defines an access point for a small set ofsweat glands such that perspiration spontaneously initiates routing ofsweat through a microfluidic network and set of reservoirs. Embeddedchemical analyses respond in colorimetric fashion to markers such aschloride and hydronium ions, glucose and lactate. Wireless interfaces todigital image capture hardware serve as a means for quantitation. Twoseparate human studies demonstrated functionality of this microfluidicdevice in different subjects during indoor fitness cycling in acontrolled environment, and also during long distance bicycle racing inarid and complex conditions. The results include quantitative values forsweat rate, total sweat loss, pH and concentration of both chloride andlactate.

A convergence of advances in materials, new concepts in mechanicsdesign, and specialized device architectures, is beginning to establishthe foundation for the next generation of wearable electronictechnologies, where sensors and other functional components reside, notin conventional rigid packages mounted on straps or bands, but insteaddirectly on the skin (1, 2). Here, we describe constructs that combinesoft, low-modulus physical properties and thin layouts allowing robust,non-irritating, and long-lived interfaces with human epidermis (2). Thisdeveloping field involves innovative ideas in both organic and inorganicfunctional materials, where mechanical and manufacturing science playimportant roles. Although most devices described in the literature focuson measurement of physical characteristics such as motion, strain,stiffness, temperature, thermal conductivity, biopotential, electricalimpedance, and related parameters (1, 3-10), complementaryinformation—often with high clinical value—could be realized throughcapture and biochemical analysis of biofluids such as sweat (11, 12).

As a representative biofluid, sweat is of particular interest owing toits relative ease of non-invasive collection and its rich content ofimportant biomarkers including electrolytes, small molecules, andproteins (13, 14). Despite the importance of sweat analysis inbiomedicine, interpreting information from sweat can be difficult due touncertainties in its relationship with other biofluids, such asinterstitial fluid and blood, and due to the lack of biomedicalappliances for direct sampling and detection of multiple biomarkerswithout evaporation (15). In situ quantitative analysis of sweat istherefore of great interest for monitoring of physiologic health status(e.g., hydration state) and for the diagnosis of disease (e.g., cysticfibrosis) (16, 17). Existing systems for whole-body sweat collectionhave been confined to the laboratory (18), where standard chemicalanalysis technologies (chromatography, mass spectroscopy,electrochemical detection) can reveal the composition of collectedsamples (19). Recent attempts to detect and collect sweat simultaneouslyhave focused on direct contact of sensors on the skin (e.g., temporarytattoo) or use of fabric or paper substrates to accumulate sweat forelectrochemical and/or optical assessment (20). For instance,electrochemical sensors directly laminated on the epidermis can detectchemical components, such as sodium ions and lactate, in real-time(21-23). Colorimetric responses in functionalized porous substrates canyield chemical information, such as the pH of sweat, and further enablesimple quantitative assays using devices capable of capturinghigh-quality digital images, such as smartphones (24-26). Radiofrequency identification (RFID) systems, which can be integrated on topof porous materials for wireless information transfer, provideadditional functionality (27, 28). These and related technologies havecome together to quantify sweat generation rate (27), but because thesweat gland density is not known, the total sweat rate and volumetricloss cannot be determined accurately in conventional technologies.Additionally, the formats do not simultaneously reveal the concentrationof multiple chemical components, nor do they offer full compatibilitywith the growing availability in soft, skin-mounted electronics,physical sensors, radio technologies, and energy storage devices.

Here, we report a type of thin and soft, closed microfluidic system thatcan directly and reliably harvest sweat from pores on the surface of theskin. The device routes this sweat to different channels and reservoirsfor multi-parametric sensing of markers of interest, with the option towirelessly interface with external devices for image capture andanalysis. This type of microfluidic technology includes fluid handling,fluid capture, and biochemical analytical capabilities. The devicescould be mounted at multiple locations on the body without chemical orphysical irritation by biocompatible adhesives and device mechanics, andformed flexible and stretchable, water-tight interfaces. These deviceswere able to measure total sweat loss, pH, lactate, chloride, andglucose concentrations by colorimetric detection using wireless datatransmission. We tested these devices in two human studies: acontrolled, indoor, mild sweat inducing study; and a “real world”,outdoor use study conducted during a long distance bicycling race.

Materials and Methods: Study design: The objectives of indoor andoutdoor human trial studies were to investigate feasibility of usingthese epidermal microfluidics devices in practical scenarios undercontrolled and uncontrolled environmental conditions and during moderateand vigorous exercise. Nine subjects were recruited through the ClinicalResearch Laboratory, LLC for indoor studies with anonymous collection ofinformation including date of birth, gender, contraceptive status,weight, height, body mass index (BMI), blood pressure, and informationfrom a simple survey of medical condition to ensure all subjects werehealthy. The experimental conditions, including temperature, humidity,time course of application of device, and weight of absorbing Webrilpads for sweat collection were all controlled and/or documented. Resultsobtained from image analysis methods (described in the section“Near-field communication and image processing for quantitativeanalysis”) were compared with those from chemical laboratory analysis.For the outdoor study, twelve healthy subjects volunteered undereligibility requirements including enrollment and participation in ElTour de Tucson, a 104-km bike race. Age, height, and weight wererecorded from subjects at the start of the race and used to calculateBMI and body surface area (BSA). Environmental conditions includingtemperature, humidity, and UV index were recorded every 2-3 hours frominformation provided by the National Weather Service. In both studies,sweat patches were placed on two different geographical body areas(volar arm and lower back) and image data were obtained by smartphoneand digital single-lens reflex (DSLR) cameras.

Fabrication of epidermal microfluidic devices with integratedelectronics for colorimetric sweat analysis: Standard soft lithographictechniques enabled fabrication of epidermal microfluidic devices (56).Briefly, casting and curing PDMS against lithographically prepared moldsyielded solid elastomers with features of relief on their surfaces.Bonding separate pieces of PDMS formed in this manner defined sealedmicrofluidic channels and containment reservoirs. Mechanical punchescreated openings to define the inlets for sweat collection. A separate,double-sided thin adhesive layer with matching holes bonded to thebottom surface of the device on one side and to the skin on the other.As an option, separately fabricated thin electronic systems with openarchitectures were mounted on the top surface. For colorimetricanalysis, the chromogenic reagents for detecting glucose, lactate,chloride, and pH were spotted onto filter paper and inserted intocontainment reservoirs. Cobalt chloride dissolved in pHEMA hydrogel wasadded to serpentine channels. Complete fabrication and colorimetricanalysis details are provided herein.

Statistical analysis: Data are presented with average values andstandard deviations (SD) unless noted in the figure caption. Significantdifference was calculated based on the two-tailed t-test. Pearson andSpearman correlation analyses were conducted on the patch and laboratoryresults (FIG. 23). The matrix of bivariate correlations in analyteconcentrations between the patch and lab analysis are displayed using aheat map representation. Blue and red denote negative and positivecorrelations, respectively. Bivariate correlations are described usingSpearman rank-order statistics. Analyses were performed using SAS andJMP statistical software.

Soft epidermal microfluidics for sweat monitoring: The soft, epidermalmicrofluidic device that we have developed adheres and conforms to theskin in a manner that captures and routes sweat through a network ofmicrochannels and reservoirs—using a combination of capillarity andaction of the natural pressure (˜70 kPa) associated withperspiration—for volumetric assessment and chemical analysis in situ(13). Low modulus biocompatible materials, soft silicon elastomers (˜1MPa), were created using soft lithography to define the microfluidicconstructs (diameter 3 cm and ˜700 μm in thickness) (FIG. 1, FIG. 7).The specific designs described can retain ˜50 μL of sweat correspondingto an effective working time of 1-6 hours of exercise, depending on therate of sweat loss and the mounting location on the body (12-120μL/h/cm²) (32). Stretchable electronics technology allows directintegration of wireless sensing and data transfer capabilities intothese platforms.

Devices are composed of a multilayer stack of three sub-systems: (1) askin-compatible adhesive layer with a micromachined opening that definesthe area of sweat collection; (2) a sealed collection of softmicrofluidic channels and reservoirs filled with color-responsivematerials for quantitative analysis of sweat volume and chemistry; and(3) a magnetic loop antenna and associated near-field communication(NFC) electronics for interfacing to external wireless devices (FIG. 1a). A medical-grade acrylic adhesive film ensured stable, strong andseamless adhesion (˜5.7 N) of the device to the skin without irritationin a manner that offered compatibility with sweaty or hairy skin (FIG.8). This adhesive exhibited ˜5 times greater adhesion force than thetypical medical adhesive Tegaderm (1.02 N) (33). The thin geometry (25μm) and low modulus (˜17 kPa) of this layer provided stress releaseduring deformation of the skin (FIG. 8) facilitating comfort andlong-term wearability. An opening defined the sweat harvesting area (3mm diameter, corresponding to ˜10 sweat glands) (34) through which sweatcould pass into the inlet region of the overlying soft microfluidicsystem (FIG. 1b ). The pressure that drives fluid flow arises from theaction of the sweat glands themselves, assisted by capillary effects inthe microchannels and the materials embedded within them. The conformalcontact of the adhesive layer inhibited lateral flow of sweat fromregions located outside the defined openings, ensuring fluid issuingfrom the harvesting area dominated the sweat sample (FIG. 9).

The microfluidic system may comprise a bottom polymer layer (e.g.,polydimethylsiloxane (PDMS)), such as having a 500 μm thickness,embossed with desired relief geometry to provide a microfluidic network,such as a uniform depth of 300 μm and filled with reagents forcolorimetric analysis (FIGS. 1a and b ). A top-capping layer of PDMSserved as a seal (200 μm thick). One layout includes four chambers (suchas circular chambers having a diameter of about 4 mm) as independentreservoirs for analysis, preventing any crosstalk, that are surroundedat the outer perimeter by an orbicular serpentine channel. This channeland each of the reservoirs are connected by separate guiding channels tothe hole segments (diameter 0.5 mm) that spatially align with theopenings (diameter 3 mm) in the skin adhesive layer (FIGS. 1b and c ).To avoid backpressure that can impede fluid flow, all channels andreservoirs may interface to an outlet microfluidic channel (100 μmwidth) that terminates on the top-side edge of the device (FIG. 1b ).Quantitative colorimetric assay reagents in the circular reservoirs inthe middle of the device facilitates assessment of pH and theconcentration of selected essential markers, including glucose, lactate,and chloride, through either enzymatic or chromogenic reactions. Assuch, with the exception of pH, the colorimetric schemes embedded in thecurrent devices do not afford real-time tracking of changes in analyteconcentration. A water-responsive chromogenic reagent in the serpentinechannel allows determination of the extent of filling with sweat, whichcan be converted to overall sweat rate and volume.

Specially formulated variants of PDMS offer physical characteristicsthat are attractive for this application, including opticaltransparency, ease of patterning into microfluidic systems,biocompatibility, and favorable mechanics (low modulus, ˜145 kPa; highelasticity, up to ˜200% strain at break) (35). The soft mechanics andthin geometry enabled soft, non-irritating intimate contact with theskin through principles similar to those established for epidermalelectronics (2, 36). The finite element analysis (FEA) results ofstrain/stress distributions and corresponding optical images in FIG. 1eshow deformation of a representative device under various mechanicaldistortions with a phantom skin (a PDMS substrate exhibiting similarmechanical properties to skin). The maximum normal and shear stresses atthe device/phantom skin interface were far below the threshold forsomatosensory perception of forces (20 kPa) during ˜30% stretch (37)(FIG. 10). Fabricated elastomeric microfluidic devices exhibited ˜0.16MPa of effective modulus, comparable to real skin and previouslyreported epidermal devices (37, 38).

Integrated electronics allow wireless interfaces to external computingand digital analysis systems using common platforms such as thesmartphone. Our technology capitalized on NFC schemes to launch imagecapture and analysis software on such an external device, and/or to readtemperature from an integrated sensor. The overall designs allowedstretchable electronics to operate under physical deformation withoutsignificantly altering the mechanical properties of the softmicrofluidic system. Finite element analysis (FEA) results demonstratedthat the maximum strain in the copper layer was below the elastic limit(0.3%) under all loading conditions (FIG. 11) (39). Reference marks onthe top of the device platform (FIG. 1b ) included a white dot and blackcrosses for color balancing to allow accurate color extraction underarbitrary lighting condition (FIG. 12). The crosses also helpeddetermine the position and orientation from the images (FIG. 13).

Soft epidermal microfluidics for sweat monitoring: We optimizedmaterials and channel design to adequately collect sweat in situ, withsoft, stretchable mechanics that offer high structure stability, lowvapor permeability, and minimal backpressure (flow impedance into thechannel). FIG. 2a is a sketch of the channel geometry, representing thearea of the outlet and the serpentine channel, used for theoreticalcalculation of the essential mechanics and flow properties. The blue andred dashed boxes highlight the dimensions of the serpentine and outletchannels, respectively. The outlet channels relieve backpressure. Theoutlet channels may yield some sweat loss as water (sweat) vapor. Thewater vapor loss showed little dependence on the length of outletchannel while backpressure was linearly proportional to this lengthaccording to calculations for a model system (FIG. 14 and FIG. 2b ). Ashort outlet channel length of 2.5 mm was chosen to minimizebackpressure. Our calculations further indicated that the vapor losswith 100 μm wide channels was ˜3.2-fold lower than with 800 μm widechannels, while 25 μm wide channels differed only ˜1.1-fold from the 100μm wide channels (FIG. 2b ). In the range of widths under 100 μm,backpressure notably increased and bending deformation of the device wasobstructed with negligible effects on vapor loss. Considering calculatedvalues and practical resolution limits of soft lithography, optimizedoutlet channel dimensions of 100 μm in width and 2.5 mm in length wereselected.

As with vapor loss and backpressure, stretchability and structuralstability are two other competing issues that demand carefuloptimization. Although thin geometries and low modulus elastomer are keyto achieving mechanical compatibility with the skin, suchcharacteristics may also yield substantial deformation or even collapseof the channel under external pressure (40), in the as-fabricated formor in states induced by natural deformations of the skin. Modelingyielded predictions for percentage changes in the volume of theserpentine channel associated with externally applied pressures between100 and 400 Pa (FIG. 2c ) comparable to those that might be associatedwith a gentle touch by a fingertip (41). The volume change increased theaspect ratio (AR, width to height). For example, the volume change forAR 5 (1.5 mm width and 300 μm height) was ˜5-fold greater than that forAR 3.3 (1.0 mm width and 300 μm height). Because the channel also needsto consider the total volume of sweat that can be captured and theoverall size of the device, we chose the lower limit AR 3.3 for thechannel design. Additionally, serpentine channel layouts provided aconvenient means to increase the total channel volume for a given devicesize (FIG. 15). All of these optimizations led to the overall design,the cross-sectional channel, and the outlet shapes illustrated in FIG. 2d.

Microfluidic sweat capture and quantitative colorimetric analysis:Quantitative in vitro testing of microfluidic performance first involveda simple, artificial sweat pore system (FIGS. 2e and f, and FIG. 16) tomimic human eccrine sweat glands (42), consisting of a perforatedpolyimide (PI) membrane (pores with diameter 60 μm, ˜100 pores/cm²)mounted in a fixture with an underlying fluid reservoir connected to asyringe pump. A device (0.07 cm² of harvesting surface area) laminatedon the perforated membrane captured dyed water pumped at 5.5 μL/hour(FIG. 2g ), demonstrating the first step in quantitative analysis ofliquid uptake. The experimentally determined harvested liquid volume inthe channel is consistent with the input volume introduced by thesyringe pump, is consistent with linear hydrodynamic flow in themicrofluidic channel, showed negligible loss of water vapor, and nofluid leakage under these conditions.

In terms of device design, three factors determine the time resolution:(1) the rate of fluid flow into the reservoirs and the serpentinechannel; (2) the harvesting area; and (3) the time and spatialresolution of the camera system and image analysis software. For thedevice layout with harvesting area (˜10 mm²), the human studiespresented subsequently showed volumetric sweat harvesting rates of˜1.2-12 μL/h, corresponding to linear filling rates of ˜0.07-0.7 mm/minalong the serpentine channels. The reservoirs would fill within ˜0.3-3.2hours at these sweating rates, with time scaling linearly with reservoirvolume. Decreasing the cross-sectional area of the channel wouldincrease the filling rate proportionally. For image capture once every 5minutes, a spatial resolution of ˜0.35-3.5 mm can easily resolve changesin the positions of the fluid fronts, providing 12 data points withinthe ˜60 min timeframe, which we considered relevant for changes in sweatchemistry (13).

The colorimetric sensing approach allowed simple, rapid quantitativeassessment of instantaneous rate and total volume and sweat loss, pH,chloride, lactate, and glucose in sweat (FIG. 3a ). The first parametersrelate to thermal regulation and dehydration, where continuousmonitoring yields important information of relevance to electrolytebalance and rehydration (43). In the orbicular serpentine channel,cobalt (II) chloride (i.e., CoCl₂) contained in a coating of apolyhydroxyethylmethacrylate hydrogel (pHEMA) matrix, served as acolorimetric indicator. As sweat entered the channel, the anhydrouscobalt (II) chloride chelated with water to form hexahydrate cobaltchloride (CoCl₂.6H₂O), generating a change in color from deep blue(λ_(max)=657) to pale purple (λ_(max)=511) (FIG. 3b ). The position ofthe leading edge that defines this color change, along with thedimensional characteristics and geometry of the channel, yieldsquantitative information on the sweat rate and volume. Owing to the thinlayer (˜25 μm) coated on the channel wall and the hydrophilic propertiesof the pHEMA hydrogel matrix, the hydrodynamics of flow within thechannel were not influenced during conditions of momentary flow (FIGS.17a-d ). The sweat could, however, continue to travel slowly through thechannel by spontaneous internal flow (0.68 μL/h), with the possibilityof a ˜2% reading error (FIGS. 17e and f ). This artifact does not occurin channels without the hydrogel; its effect could be determined, forpractical purposes, by patterning the hydrogel into short segments (FIG.18).

Four different paper-based colorimetric chemical assays resided in thecentral reservoirs. The cellulose matrices in each reservoir could befilled with as little as 5-10 μL of sweat sample. The color changesoccurred on timescales of <1 min. The concentration of lactate in sweatis an indicator of exercise intolerance and tissue hypoxia (44, 45).Enzymatic reactions between lactate and co-factor NAD+ by lactatedehydrogenase and diaphorase induce a change in color of a chromogenicreagent (i.e., Formazan dyes). The formulation of enzyme and dyes in thedetection cocktail solution ensured a dynamic range compatible withhuman sweat. The color change in the detection reservoir correlated withthe concentration of lactate throughout the relevant range expected insweat (1.5-100 mM) (FIG. 3c ) (13, 46, 47).

Glucose concentration could also be analyzed by an enzymatic reaction(FIG. 3d ). Glucose oxidase physically immobilized in a cellulose matrixproduces hydrogen peroxide associated with oxidation of glucose andreduction of oxygen. Following this reaction, iodide oxidizes to iodineby peroxidase, to yield a change in color from yellow (iodide) to brown(iodine), to an extent defined by the concentration of glucose (48, 49).We note that glucose concentration in sweat is typically one order ofmagnitude lower than in plasma; the range of sensitivity in the reporteddevices could diagnose hyperglycemia, for example [limit of detection(LOD)=˜200 μM] (FIG. 19) (50). Further development of colorimetricchemistries based on enzymatic reactions and/or enzyme-mimeticnanomaterials could improve the limits of detection (52, 53). Similarly,creatinine, a vital marker of hydration status and renal function, wasdetected in sweat using a mixture of enzymes (creatininase, creatinase,and peroxidase) and a corresponding responsive dye (4-amino phenazone)(FIG. 3e ) (51).

In sweat, pH is often considered an index of hydration state; theconcentration of chloride ions serves as a marker of cystic fibrosis;and altered electrolyte levels correspond to a sodium ion imbalance(17). A universal pH indicator that includes dyes such as bromothymolblue, methyl red, and phenolphthalein yielded colorimetric responsesover a medically relevant range (pH 5.0-7.0) (FIG. 3f ). Colorimetricdetection of chloride involved competitive binding between Hg²⁺ and Fe²⁺with 2,4,6-Tris(2-pyridiyl)-s-triazine (TPTZ). In the presence ofchloride ions, iron ions (Fe²⁺) bind with TPTZ while Hg2+ participatesas HgCl₂, thereby inducing a change in color from transparent to blue asshown in FIG. 3 g. Although PDMS is known to have some permeability towater and certain small molecules, the colorimetric responses in thesedevices are not influenced for most practical applications due to therelevant operational timescale and the analyte chemistries used (FIG.20).

Near field communication interface to a smartphone and image processing:Recording color changes and converting them into quantitativeinformation was accomplished by digital image capture and analysis. FIG.4a shows frames from a video clip in which the proximity of a smartphoneto the device initiated image capture and analysis softwareautomatically using NFC. The user then adjusted the viewing position tothe targeted spot to determine exact RGB color in situ. The applicationdigitized RGB color information on the screen, enabling the user to readthe concentration of the marker. The previously reported ultrathin NFCelectronics (39) integrated on the top of the microfluidic deviceenabled wireless communication to external devices, with stableoperation and a soft, biocompatible set of mechanical properties, evenunder a 30% strain condition (39). The NFC electronics facilitated imagecapture, and built-in temperature sensors on the NFC chips providedwireless, digital data on skin temperature.

After wirelessly collecting images, image processing for assessment ofcolor changes was achieved as shown in FIGS. 4b -d. Reference colormarkers (true white and black) allowed white balancing to eliminate thedependence of the analysis on lighting conditions of practical relevance(daylight, shadow, and various light sources) (FIG. 12). In particular,a white dot in the middle of the device and four black crossesdistributed near the center established values for 100% and 0% in % RGBcoordinates, respectively (FIG. 4b ). The crosses further allowedrotations/translations of the images to facilitate accurate analysis ofsweat rate and volume on the serpentine channel (FIG. 13). After imagecorrection, the digital color data (in % RGB format) were converted intoanalyte concentrations using calibration curves (FIG. 4d ). We couldreliably measure changes of 0.5 pH units and 0.2, 0.3, and 0.1 mM ofchloride, lactate, and glucose concentrations, respectively,corresponding to a 1% change in the R channel of the RGB images.Although the calibration curve in FIG. 4c captures the non-linearitiesassociated with the serpentine shape of the channel, the angle of thefilling front (the leading edge of the color change) in the serpentinechannel defined the volume of sweat collected, thereby allowingcalculation of total sweat loss and, with the time interval, total sweatrate.

Human testing of the skin-mounted sweat sensor: The first demonstrationof practical utility involved nine human subjects with the devicemounted on two different body locations (lower back and volar forearm,FIG. 5b ) and with two different harvesting areas (size of the openingin the adhesive shown in FIG. 5a ) during intermediate-level activity oncycle ergometers under controlled 38° C. temperature and 50% relativehumidity conditions. We compared the performance of the device in situto conventional procedures that use absorbing pads applied onto the skinwith subsequent weighing and lab-based analyses, such asspectrophotometry.

We quantified regional sweat rate normalized to unit area over thecourse of 1 hour (FIGS. 5c and d ). Although the rates exhibited greatvariation among individual subjects, sweat rates measured on the lowerback were typically ˜2.3-fold greater than on the volar forearm,consistent with expectations from studies using conventional techniques(54). Rates determined using the devices with large harvesting areasshowed agreement with those obtained using absorbing pads (FIG. 5e ).Furthermore, the devices accurately captured the volume and rateinformation continuously, without the need for removal. Notably, they-intercept in FIG. 5e corresponds to the limit of sweat measurementwith the absorbing pad due to water evaporation (0.349 g/cm²s of waterevaporation at 38° C., 50% relative humidity) during sample collection,highlighting one of its limitations. Devices with small harvesting areasyielded somewhat higher inferred rates than those with larger harvestingareas, perhaps due to alternations in perspiration behavior caused bythe physical presence of the device (FIG. 5e ) (55).

Concentrations of the markers chloride, glucose, lactate, and pHobtained by the colorimetric readouts demonstrated excellent agreementwith conventional laboratory analysis of sweat collected from absorbingpads as shown in FIG. 5 f. The glucose concentration in sweat fromhealthy subjects fell below the limit of detection for both image andlab analysis (p<0.05 refers to differences in background noise only).Bivariate and multivariate statistical analyses, together with Pearsoncorrelation heat maps and Spearman rank-order statistics, quantified thecorrelations for all markers tested (FIG. 23).

To examine the mechanical and fluidic integrity of the devices in ademanding exercise scenario, we assessed robustness in adhesion andfluidic collection and capture using devices on volunteers in acompetitive long distance outdoor bicycling race—El Tour de Tucson.Testing involved patch placement on the lower back and the volar surfaceof the forearm of 12 volunteer riders (FIG. 6b ). In all cases, thedevices performed as anticipated, successfully collecting sweat withregional colorimetric change without patch detachment, even withsubstantial changes in temperature and humidity. Participants reportedno sense of discomfort or limitation in body or arm movement during thecycling. Older subjects (ages 50-69) had greater rates of sweating incomparison to that of younger subjects (ages 10-29), and male subjectsexhibited greater rates of sweating than females (FIG. 6e ).

The epidermal microfluidic devices introduced herein represent versatileplatforms for evaluating athletic performance and monitoring health anddisease status. The devices may detect sweat volume and rate, as well asseveral key markers including glucose, creatinine, lactate, chloride,and pH. Compared to previously described technologies for sweat analysisconsisting of porous materials and fabrics or hydrogels as fluidicinterfaces, our systems are unique in their use of fully integrated,soft microfluidics consisting of a network of functionalized channelsand reservoirs for sweat capture, routing, and storage with spatiallyseparated regions for analysis. The devices herein may provide furtherquantitative modes of use for additional applications.

In addition to systematic investigations of the key engineering aspectsand design parameters, initial studies demonstrated practical utilitythrough tests on nine volunteers during moderate intensity exercise incontrolled conditions, with correlation of measured results to standardmethods based on absorbent pads and laboratory chemical analyses.Evaluations on twelve cyclists during high intensity physical exertionrevealed real-word performance without loss of adhesion, leakage offluids or other modes of failure, and without discomfort or irritationat the device/skin interface.

The soft mechanical properties, biocompatible constituent materials,digitally analyzable colorimetric responses, and overall carefuloptimization of structural, evaporative and fluidic properties areintegral to the effectiveness of these devices and differentiate themfrom other sweat analysis devices. The applications include use of thesedevices for real-time, in situ sweat analysis and as storage vehiclesfor ex situ laboratory evaluation. In this latter context, it isimportant to note that we observed that the microfluidics structuresdescribed here can hold captured sweat for ˜125 hours upon removal fromthe skin and sealing of the open channels (˜75 hours without sealing)with negligible deterioration of colorimetric analysis.

These colorimetric schemes may be extended to include enzymaticreactions or chromogens aimed at a broad range of possible applicationsfor specific clinical diagnosis or for illicit drug use detection.Advanced electronic or non-electronic strategies for temporal trackingof sweat chemistry are of interest. An alternative approach is inmicrofluidic designs that enable time-dependent sampling of sweat intospatially distinct reservoirs for separate analysis. In all cases,digital image capture analysis represents a simple, ‘wireless’ means ofquantitation. Direct electronic readout represents an additionalpossibility, where epidermal power supplies or wireless power transferschemes could be useful.

In addition to their use in sweat monitoring, similar systems are usableas direct capture and storage vehicles for subsequent colorimetric orconventional lab-based analysis for various accumulated biofluids suchas tears, saliva, or discharges from wounds, especially for small samplevolume collection (<˜50 μL). The same platforms are combinable withelectronic or pharmacological means to actively initiate the release ofsweat or extraction of other biofluids (e.g., interstitial fluids). Inboth such active and passive collection modes, the devices are usable inathletic and military training to gain insight into critical electrolyteloss, thereby guiding earlier supplementation before symptomaticcramping and ‘hitting the wall’ points in time at which appropriatepreventative treatment is no longer effective. In this scenario, and inothers of interest, data accumulated over time from individual users isusable as the basis for the development of analytic approaches forinterpreting trends in marker concentrations, with the potential toprovide warning signs associated with physical activities that lead toabnormal responses. The intrinsically simple, low-cost nature of thedevices facilitates rapid, broad distribution for use in these contexts.

Fabrication of epidermal microfluidic devices with integratedelectronics: Photolithography using a negative photoresist SPR 220 4.5(MicroChem) and deep reactive-ion etching (STS Pegasus ICP-DRIE, SPTSTechnologies Ltd) generated topographically defined channels (300 μm indepth) on a silicon wafer, as a master. A thin, spin-cast layer ofpolymethyl methacrylate (PMMA; 3000 rpm for 30 s following with curingat 180° C. for 5 min) prevented adhesion of a layer ofpolydimethylsiloxane (PDMS; Sylgard 184, Dow Corning; mixed at a 30:1ratio of base to curing agent by weight) cast at 200 rpm for 30 s andcured at 70° C. for 4 h. This process yielded an inverse replica of therelief on the silicon in a slab of PDMS (˜700 μm total thickness). Thecolorimetric dyes were then loaded by fill-and-dry and drop-castingmethod in the channels and reservoirs, respectively. Details ofpreparation of detection cocktails are listed below.

The electronic part of the system consisted of a thin device with open,deformable architecture and near field communication (NFC) capabilities,constructed according to procedures described elsewhere (39).Electrodeposition of the backside of this system with SiO₂ allowedcovalent bonding to a film of PDMS (˜200 μm thickness) through chemicalinteractions where —OH groups formed by exposure to oxygen plasma. Asimilar bonding strategy joined this sub-system to the molded PDMS, toyield a network of closed microfluidic channels. A final bonding stepintegrating a layer of skin adhesive (PC2723U, ScapaHealthcare) alsoused a same condition. All plasma bonding steps used an oxygen plasmaequipment (Harrick Plasma Cleaner, Harrick Plasma) under pressure of 500mTorr with ‘high’ power for 1 min. A custom temporary tattoo laminatedon the top of device provided alignment marks and color calibrationfeatures for image processing. The devices were stored at 4° C. in arefrigerator with food-saver sealing to preserve the enzymes and NAD+.

Preparation for quantitative colorimetric analysis of biomarkers: Theserpentine microfluidic channels were coated with a solution of 100mg/mL cobalt (II) chloride dissolved in a 2 wt %polyhydroxyethylmethacrylate (pHEMA) hydrogel. The resulting bluematerial formed thin layers on the internal walls of the channels afterdrying at ambient conditions for 30 min. Colorimetric analysis reagentswere spotted on filter paper (i.e., 4 mm dia.) in their respective testzones. A 2 μL volume of a universal pH indicator solution (RiccaChemical Company) served as a chromogen for pH detection. Preparation ofthe glucose detection cocktail involved dissolving 1.2 mg of glucoseoxidase, 0.12 mg of horseradish peroxidase, 102 mg of trehalose, and99.6 mg of potassium iodide into 1 mL of sodium citrate buffer solution(pH 6.0) (48, 49). A small volume (i.e., 5 μL) of this cocktail wasintroduced into the glucose detection reservoir. The lactate assayreservoir was prepared by adding a 3 μL mixture of lactate enzyme,substrate solution, and assay buffer (D-Lactase Assay Kit;Sigma-Aldrich) in a ratio of 3:2:5, respectively. A chloride detectionreagent (3 μL; Chloride Assay Kit, Sigma-Aldrich) titrated with 0.1 wt %HgSCN until a clear appearance provided the functional component of thechloride assay reservoir. All of these paper-based assays were allowedto dry before physically inserted into the four circular reservoirsregions.

Optical absorption spectra in the UV-Vis range (i.e., 400-700 nm)collected using a double-beam Cary 5G spectrophotometer (Varian) withdisk-shaped specimen holders (2 mm dia.) provided data to calibrate thevarious colorimetric responses. Test samples consisted of 5 μL ofstandard solutions with various concentrations placed on the paper-basedassay substrates. Bare filter paper served as a reference material.These experimental samples were also used for constructing calibrationcurves of image processing.

Assessment of an epidermal microfluidic device: Scanning electronmicroscope (SEM; HITACHI S-4700, Hitachi) images were collected at anaccelerating voltage of 5 kV after sputter-coating a thin layer of gold(20 nm). Mechanical properties were assessed with a DMA 800 (TAinstruments) using a single cantilever for the rectangular shape blockprepared by cutting the device. An artificial sweat simulator enabled invitro evaluation of the device performance by mimicking the humanperspiration system. The simulator consisted of a membrane with an arrayof pores prepared by laser drilling through a polyimide membrane (poreswith 60 μm diameters at a density of 100 pores/cm² on membranes with 50μm thickness). The pore-containing membrane was mounted onto a fluidchamber connected to a syringe pump while feeding water containing bluefood dye with 5.5 μL/hour input rate for 6 hours (42).

Finite element analysis (FEA) and mechanics model of the microfluidicstructures: ABAQUS commercial software (Dassault Systems) was used forFEA of the device under different external loads (stretching, bendingand twisting). The focuses are to ensure that (1) the interfacial normaland shear stresses below the low somatosensory perception of the deviceon the human skin; and (2) the strain in the copper layer of the NFCelectronics below the elastic limit such that no plastic yieldingoccurs. The classic theory for plates was used to calculate thedeflection of the cover layer and volume change of the microfluidicchannel under uniform in-plane pressure. An analytic model wasestablished to quantitatively estimate the backpressure induced by inletsweat flow for different outlet width. Simulation details and materialproperties (e.g., Young's modulus) are described below.

Full 3D FEA was used to study the mechanical performance of the devices,which were mounted on skin (100×50×2 mm³) subjected to stretching,bending and twisting. For stretching, displacements corresponding to 30%stretching were applied to two ends of the skin, which resulted ˜16.8%average tensile strain on the bottom surface of the device along thestretching direction. For twisting, each end of the skin was subjectedto 90-degree rotation such that the two ends of the skin had a180-degree rotation. For bending, the displacement field applied to thebottom surface of the skin corresponded to 2 cm bending radius ofcurvature. Eight-node 3D solid elements were used for skin and PDMSmicrofluidic system, while four-node shell elements were used for theNFC electronics.

The elastic modulus and Poisson's ratio used in the simulations are 0.06MPa and 0.49 for the skin (37); 0.145 MPa and 0.49 for the PDMS of themicrofluidic system; 0.017 MP and 0.49 for the skin adhesive; 119 GPaand 0.34 for copper in the NFC coil; and 2.5 GPa and 0.34 for the PI ofthe NFC coil encapsulation.

Deformation under Uniform Pressure: The cross-section of the serpentinechannel is shown in FIG. 2a and FIG. 21. The cover layer of the channelis modelled as a shell under cylindrical bending with clamped boundaryconditions. For a channel of width under uniform pressure, the maximumdeflection of the cover layer is (57)

$\begin{matrix}{w_{\max} = \frac{pa^{4}}{384\overset{¯}{E}I}} & ({II})\end{matrix}$

Here

${\overset{¯}{E}I} = {\frac{E}{1 - \nu^{2}}\frac{t^{3}}{12}}$

is the bending stiffness, where E, v are Young's modulus and Poisson'sratio of the elastomer, respectively, and t is the thickness of thecover layer. The percentage change of volume can be calculated byintegrating the deflection along the cover layer as

$\begin{matrix}{\frac{\Delta V}{V_{0}} = {\frac{pa^{4}}{720\overset{¯}{E}{Ih}} = {\frac{ph^{3}}{720\overset{¯}{E}I}\left( \frac{a}{h} \right)^{4}}}} & ({III})\end{matrix}$

It scales with the fourth power of the channel width a, and thereforecan be drastically decreased as a decreases (FIG. 2c ).

Analysis of Backpressure: For the air inside the microfluidic channel(FIG. 21a ), its volume V decreases as sweat flows into the channel, andsatisfies the ideal gas law

PV=nRT   (IV)

where P, T and n are the pressure, temperature and amount of substance(number of moles), respectively, and R is the ideal gas constant. It isimportant for the microfluidic channel to have an outlet, without whichthe pressure P would continue to increase as V decreases, and wouldimpede the fluid flow. For a constant temperature, the rate form of Eq.V gives

{dot over (P)}V+P{dot over (V)}={dot over (n)}RT   (V)

Here {dot over (V)}=−{dot over (V)}_(inlet) for an incompressible fluid,where {dot over (V)}_(inlet) is the inlet sweat flow rate. For aconstant {dot over (V)}_(inlet) and sufficiently long time such that Preaches a steady-state value (and {dot over (P)} approaches zero), theair escape rate {dot over (n)} is given by

$\begin{matrix}{\overset{.}{n} = {- \frac{P{\overset{.}{V}}_{inlet}}{RT}}} & ({VI})\end{matrix}$

Another relation between {dot over (n)} and pressure P can be obtainedfrom the air flow through outlet channel, which is modelled as arectangular tube of length L, height h and width w (FIGS. 21b and c ).The mean velocity ū of air through the outlet is linearly proportionalto the pressure difference on both sides of the outlet channel ΔP=P−P₀(P₀ is the atmosphere pressure) (58)

$\begin{matrix}{\overset{¯}{u} = \frac{\left( \frac{2wh}{w + h} \right)^{2}\Delta P}{32\mu L}} & ({VII})\end{matrix}$

where μ is the viscosity of the air. The air escape rate {dot over (n)}is related to ū by

${\overset{.}{n} = {- \frac{\overset{¯}{u}wh\rho}{M_{air}}}},$

where ρ and M_(air) are the density and molar mass of the air,respectively. The relation between {dot over (n)} and ΔP is then givenas

$\begin{matrix}{\overset{.}{n} = {{- \frac{{\left( \frac{2wh}{w + h} \right)^{2}{hw}\; \rho}\;}{32\mu LM_{air}}}\Delta P}} & ({VIII})\end{matrix}$

Elimination of from Eq. VI and Eq. VII gives backpressure ΔP

$\begin{matrix}{{{\Delta P} = {\frac{P_{0}}{{\frac{w^{3}h^{3}}{8{L\left( {w + h} \right)}^{2}}\frac{\rho}{\mu \; M_{air}}\frac{RT}{{\overset{.}{V}}_{inlet}}} - 1} \approx \frac{8{L\left( {w + h} \right)}^{2}}{w^{3}h^{3}}}}\frac{\mu \; M_{air}}{\rho}\frac{{\overset{.}{V}}_{inlet}P_{0}}{RT}} & ({IX})\end{matrix}$

The constants used in all calculations include the atmosphere pressureP₀=10⁵ Pa; temperature T=300K; molar mass M_(air)=29.0 kg/mol, viscosityμ=1.8×10⁻⁵ Pa·s, and density ρ=1.2 kg/m³ of air; idea gas constantR=8.31 J·mol⁻¹·K⁻¹; and the sweat flow rate {dot over (V)}_(inlet)=15μL/hour, which corresponds to the maximum sweat rate 150 μL/(hour·cm²)(32) multiplied by the device inlet area (0.125 m²).

Near-field communication and image processing for quantitative analysis:The NFC electronics allowed communication with NFC enabled smartphones.This wireless interface automatically launched open color analysisapplication software to yield RGB values that can be connected to theconcentrations of the selected biomarkers. Image-processing algorithmsallowed quantitative analysis. Black crosses at the four corners and awhite circle in the center yielded reference colors for white balancing,by defining 1 and 0 for all RGB values, respectively. Image analysisperformed in this manner enabled extraction of accurate RGB colorcomponents, independent of lighting conditions. Known concentrations ofbiomarkers resulted in color changes to establish standard calibrationcurves. The black crosses facilitated image positioning, for reliabledetection of the angular position of the leading edge of color change inthe serpentine channel.

Human studies: In the controlled indoor study, volunteers consisted ofhealthy individuals (n=9, males and females, ages 18-40) with healthyintact skin in the investigational areas; systolic blood pressure 90-130mmHg; diastolic blood pressure 50-90 mmHg; and a Body Mass Index (BMI)between 20-30. Clinical Research Laboratories, Inc. (NJ, USA) recruitedvolunteers and the Allendale Institutional Review Board approved theprotocol (No. ACR/SWET/15-0121). Subjects provided written informedconsent. The four investigational areas included the right and leftvolar forearm and the right and left lower back. The investigationalareas were gently cleaned with soap and water, and dried beforeapplication of the devices. Two sets of microfluidic patches (a set ofsweat patches includes small and large inlet sweat devices (n=2)) wereapplied on the right lower back while one set of microfluidic patcheswere applied to the right and left for each volar forearm. A total ofeight sweat patches were thus laminated on each subject. One Webril®Handi-Pad applied to the left lower back as a reference absorbing pad.Evaluation consisted of 60 minutes of cycling, sitting, and imagecapture intervals. Subjects sat for 5 minutes first and then peddled ona bicycle ergometer for 5 minutes (22.5±1.6 km/h). After the cycling wascompleted, one image of each investigational area (lower back, right andleft forearm) was captured with an iPhone camera, an Android camera, anda Cannon camera, in standard lighting conditions. This procedurecontinued for 60 minutes in a conditioned room at 38±2° C. and 50±5%humidity. The subjects were provided water during the intervals asneeded. After the tests, the microfluidic patches and the Webril®Handi-Pad were removed and the pad were stored in a freezer (−80° C.)until time of analysis

For the outdoor real life demonstration, twelve volunteers, providinginformed consent, participating in the El Tour de Tucson (Tucson, Ariz.)perimeter bike race were enlisted. The volunteers consisted of healthyindividuals (10 Males and 2 females; ages ranging from 23-70 years; withBMI 20-33; and a body surface area (BSA) between 1.6-2.3 m²).Temperature during the El Tour inclined from 7 at the start of the raceto 25.6° C., while humidity dropped from 47 to 19% during the race.Nine, two, and one volunteers completed 167, 88, and 64 km of racing,respectively, with a collective average speed of 26.9 km/h. Allvolunteers wore the sweat monitoring patches on their lower back andvolar forearms. Before, during and after the race, sweat patch imageswere collected via a digital camera and additional sweat patches wereapplied at the mid-point of race (i.e., ˜84 km) in selected individuals.Images were obtained immediately after the race to avoid any artifactsdue to reabsorption of sweat.

Lab-based Sweat Analysis: Sweat obtained from subjects during the indoorhuman study was collected as infranatant (i.e., lower liquid portion) bycentrifuging at 2,500×g for 2 min using a tube divided with 3D printedporous barrier layer. Absorbance microplate reader examined chemicalcompositions (i.e., glucose, chloride, and lactate) of the collectedsweat (50 μL for each well) in the lab using a standard colorimetricanalysis kit (Sigma-Aldrich). The pH value was determined using a micropH meter (Hanna Instruments).

Statistical Analysis: Obtained data is presented with average value andstandard deviation (SD). Pearson and Spearman correlation analyses wereconducted on the patch and laboratory results (FIG. 23). The matrix ofbivariate correlations in biomarker concentrations between patch and labanalysis has been displayed using a heat map representation. Bluedenotes negative correlation and red positive correlation. Bivariatecorrelations have also been described using Spearman rank-orderstatistics. Analyses have been performed using SAS and JMP statisticalsoftware.

EXAMPLE 2 Thin, Soft, Skin-Mounted Microfluidic Networks with CapillaryBursting Valves for Chrono-Sampling of Sweat

Systems for time sequential capture of microliter volumes of sweatreleased from targeted regions of the skin offer the potential to enableanalysis of temporal variations in electrolyte balance and biomarkerconcentration throughout a period of interest. Conventional methods thatrely on absorbent pads taped to the skin do not offer the ease of use orthe fidelity in sweat capture needed for quantitative tracking inrealistic settings; emerging classes of electronic wearable sweatanalysis systems do not directly manage or exploit sweat-induced fluidflows for sample isolation. Here, we introduce a thin, soft, ‘skin-like’microfluidic platform that bonds to the skin to allow for collection andstorage of sweat in a set of microreservoirs. Filling occurs pressureinduced by the glands themselves to drive flow through a network ofmicrochannels that incorporate capillary bursting valves designed toburst at different pressures, for the purpose of passively guiding sweatthrough the system in a well-defined, time-ordered fashion. Theoperation is robust to mechanical stresses encountered duringapplication, operation and removal from the skin, with negligiblechemical contamination or unwanted fluid mixing. After use, a set ofcollection chambers located in a radial array at the periphery of thedevice can be filled by centrifugal force, with capability for long orshort term sample storage prior to ex situ chemical analysis. Thematerials and fabrication schemes support a broad range of choices inlayouts, sizes and numbers of microchannels and microreservoirs, fordifferent use scenarios and mounting locations. Human studiesdemonstrate applications in the accurate chemical analysis of lactate,sodium and potassium concentrations and their temporal variations.

Sweat, a biofluid excreted from eccrine glands in the epidermis,contains electrolytes (sodium, chloride) and lactate, urea and smallconcentrations of proteins, peptides and metal ions.[1] Theconcentrations of these and other biomarkers can provide importantinformation on physiological state, such as dehydration[2], and ondiseases such as cystic fibrosis[3] and childhood pancreatic disease.[4]Temporal changes and variations in the chemistry of sweat across bodypositions offer additional valuable insights into health status.[5-7] Inthis context, wearable devices capable of collecting and storing sweatinto discrete chambers have potential value. Established technologiesrely on absorbent patches (PharmChek®[8]) or coiled tubes (Macroduct®[9]), and serve only as passive vehicles for sweat collection over acertain period of time. Capturing samples at different times requiresrepeated application and removal of such devices [5-7]. Emerging formsof wearable, electronic sweat analysis systems [12-19] exploitelectrochemical approaches for monitoring biomarker concentrations, butthey also do not allow for collection, capture or subsequent analysis ofdiscrete samples of sweat captured at well-defined time points.

Recent work on thin, soft, skin-mounted microfluidic systems establishesroutes for exploiting sophisticated concepts in lab-on-a-chiptechnologies for sweat collection and analysis [10-17]. Here, sweatglands, which create pressure due to natural differences of osmolalitybetween plasma and sweat [18], actively drive flow into a network ofmicrochannels and microreservoirs. The maximum pressures generated inthis manner are estimated to be ˜70 kPa per gland, sufficient for thispurpose[19]. Although previously reported systems do not incorporate anyvalves for controlling the direction of flow through the microfluidicnetwork, piezoelectric[20], electrokinetics[21] and chemical [22]approaches are compatible with the basic platform, and can be consideredfor this purpose. Herein, we report an approach that guides flow inthese type of skin-mounted microfluidic devices via a collection ofcarefully designed capillary bursting valves (CBVs) that direct the flowof sweat to fill a collection of microreservoirs in a time sequentialmanner, thereby providing a precise sampling capability. Past work onconventional lab-on-a-chip technologies demonstrates that CBVs are usedfor stop valves [23-26] and flow guides [27, 28], but not for the typeof control achieved here. Systematic in vitro tests illustrate robust,stable function in various conformal, skin-compatible designs thatadditionally allow efficient means for storage and final extraction ofdiscrete samples of sweat. Human field testing validates the utility ofplatforms configured for sequential sweat sampling followed byextraction and ex situ chemical analysis, with a focus on lactate,sodium and potassium. Results indicated differences between sweatgenerated by thermal exposures and by running exercises, as well asvariations with position across the body.

Experimental Section: Materials and Methods: Device Fabrication:Fabrication of molds began with spin coating of a 15 μm thick film ofphotoresist (KMPR 1010) on a silicon wafer (FIG. 29). Afterphotolithography and developing, deep reactive ion etching (STS PegasusICP-DRIE, SPTS Technologies Ltd) created trenches in the silicon to adepth of 300 μm. Spin coating forms thin layer ofpoly(methylmethacrylate) (PMMA; Microchem, MA, United States) on theresulting mold. Pouring poly(dimethylsiloxane) (PDMS at a 30:1; Sylgard184, Dow corning, MI, United States) onto the PMMA film and then spincoating at 200 RPM formed a thin (400 μm) layer. A mechanical punch tooldefined holes with ˜1 mm diameters at the outlet hole of each of theextraction chambers. For capping layer, pouring PDMS (30:1) ontopolystyrene petri dish (VWR, IL, United States) and then spin coating at400 RPM formed bare layer (200 μm). A mechanical punch tool defined holewith 1 mm diameter located at the center of the capping layer. Exposureto oxygen plasma generated at low (6.8 W) RF power at 500 mTorr (PlasmaCleaner PDC-32G, Harrick Plasma, NY, United States) for 10 sec, thechannel part and capping layer are aligned for inlet and bonded. Agingthe bonded structure for 24 hours allowed the PDMS surfaces to recovertheir hydrophobic properties. Bonding to a skin adhesive (PC2723U,ScapaHealthcare) with a 2 mm diameter hole aligned to the inletcompleted the fabrication. Introduction of a solution of 100 mg/mLcobalt (II) chloride dissolved in a 2 wt % polyhydroxyethylmethacrylate(pHEMA) hydrogel (Sigma-Aldrich, MO, United States) into the channelsfacilitated visualization of the filling process.

In vitro chrono-sampling test and measurement of bursting pressure: Ahydrostatic pressure generator served as the basis for a simple in vitromodel of sweat generation, for the purpose of characterizing the CBVs(FIG. 30a ). The height of the top of a water column at the burstingpoint provided an estimate of the BP. As calibration, the pressurecreated by the generator was compared to the value from a microfluidicpressure controller (Fluigent MFCS, Villejuif, France).

In situ chrono-sampling test: Application of ethanol swabs cleaned theskin of volunteers involved in the studies, shortly before applicationof the devices. For the thermal exposure tests, the subjects remained ina dry sauna at 55° C. for 30-40 min. For the running exercises, thesubjects ran with a constant speed at ˜10 km/h. After the tests, thedevices were peeled from the skin and centrifuged at 5000 RPM to movesweat from the collection chambers into corresponding extractionchambers. For measuring sweat rate by conventional method, hydrophilicfoam dressing (Covidien, MA, United states) in 2 cm×2 cm size was used.

Chemical contamination test: Tests for chemical contamination of sweatby any of the materials used in the device construction materials used aPDMS channel 2 mm in width and 0.5 mm in depth with PDMS cover andadhesive which an overall construction identical to that of the actualdevices (FIG. 30a ). Artificial sweat consisted of an aqueous solutionof 22 mM of urea, 2.2 mM of glucose, 3.8 mM of potassium, 31 mM ofsodium, 58 mM of chloride and 5.2 mM of calcium (Sigma-Aldrich, MO,United States). This fluid filled the PDMS channel and remained therefor 2 hours at room temperature prior to recovery for chemical analysis.A similar sample, without exposure to the test structure, served as acontrol.

Chemical analysis: Analysis of lactate involved 1 μl of sweat extractedfrom the device and subsequently diluted in 100 μl of water. This samplewas introduced into liquid chromatography-mass spectrometry system(Waters Synapt G2-Si ESI, MA, United States) with an ACQUITY UPLC BEHC18 column (130 Å, 1.7 μm, 2.1 mm×50 mm) at a flow rate of 0.2 ml/min.Solvent A was 95% water, 5% acetonitrile and 0.1% formic acid; solvent Bwas 95% acetonitrile, 5% water and 0.1% formic acid. For sodium andpotassium analysis, 0.5 μl of sweat were extracted from the device andsubsequently diluted in 1 ml of water. The sample and three standardsamples were diluted in 2 ml of 0.5% nitric acid as preparation forinductively coupled plasma mass spectrometry (ICP/MS; SCIEX ELAN DRCe,PerkinElmer, CT, United States). For chloride analysis, 1 ml ofartificial sweat diluted in 25 mL of water and the 20 ml of solution wasmixed with 0.4 mL of ionic strength adjuster. Ion selective electrodewas used for measuring chloride concentration (Thermo Scientific, MA,United States).

Contact angle measurement: Static contact angles were measured using 5μl droplets of de-ionized water dispensed using an automated system andmeasured with a contact angle goniometer (KSV CAM200, Stockholm,Sweden). Contact angles were evaluated after 10 s of contact. Theresults establish the kinetics of recovery of hydrophobic properties ofthe PDMS surface.

Thin, soft microfluidic devices for chrono-sampling of sweat: The thin,soft physical properties of these devices allows their intimate,comfortable bonding to the skin for the purpose of collecting,manipulating, analyzing and storing sweat, captured in a sequentialmanner. A representative device shown in FIG. 24a has a circular overallgeometry with a diameter of 3 cm. The radial construction facilitatesthe use of centrifugation techniques for collection of sweat afterremoving the device from the skin, as described subsequently. The designinvolves two layers of poly(dimethylsiloxane) (PDMS) supported on amedical-grade acrylic adhesive film for bonding to the skin. The firstlayer defines a network of microfluidic channels (400 μm thickness;channel widths and heights are 200 μm and 300 μm, respectively) and theCBVs (designs described next), the second serves as a capping layer (200μm thickness) and the third (50 μm thickness) establishes adhesion tothe skin and defines openings (2 mm diameter) from which sweat entersthe microfluidic system (1 mm diameter, inlet; FIGS. 24b and 29). Thesystem in FIG. 24 consists of a network of microfluidic channels thatconnect to 12 separate chambers in parallel by bridging channels (FIG.24c ). Each chamber has an outlet opening (0.5 mm diameter) to releaseair pressure that would otherwise build during the filling process.

In vitro tests using dyed water illustrate the clockwise flow throughthis network (FIG. 24d ). PDMS is a good choice due to its dimensionalstability in water [29], materials biocompatibility[30], low modulus,elastic mechanical properties, and compatibility with simple molding andbonding processes for fabrication[31-33]. Careful testing indicates anabsence of chemical contamination from the PDMS and the adhesive layerin analysis of biomarkers of interest in sweat (FIG. 30). These sameresults suggest a minor (˜10%) decrease in glucose concentration,possibly due to slight absorption into the constituent materials of thedevice.

Principle and Design of the Capillary Bursting Valves for TimeSequential Sampling: The CBVs block flows at pressures lower than theircharacteristic bursting pressures (BPs) [34]. When liquid in a singleconnected channel encounters two separate CBVs with different BPs, atsufficient pressures, the flow will proceed first through the valve withlower BP. In this way, locating two CBVs with different BPs near theintersection between two channels allows control of the direction offlow. The Young-Laplace equation gives the BP in a rectangular channelas equation (Eq.) (X) [34, 35],

$\begin{matrix}{{{BP} = {{- 2}{\sigma \left\lbrack {\frac{\cos \; \theta_{I}^{*}}{b} + \frac{cos\theta_{A}}{h}} \right\rbrack}}},} & (X)\end{matrix}$

where σ is the surface tension of liquid, θ_(A) is the contact angle ofthe channel, θ*_(I) is the min[θ_(A)+β, 180°], β is diverging angle ofthe channel, b and h are width and height of the diverging section,respectively. For hydrophobic materials at high diverging angles, the BPincreases with decreasing b and h. Each unit cell of the devicesdescribed here includes three CBVs, a collection chamber, an extractionchamber and a sampling outlet (FIG. 25a ). In one embodiment, the firsttwo CBVs, denoted #1 and #2, have diverging angles of 36° and 90°,respectively, and widths of 200 μm. The third CBV, i.e. #3, has adiverging angle of 120° and a width of 50 μm (FIG. 25b ). The heights ofthe valves are 300 μm. According to Eq. (I), the contact angle of thechannel surfaces affects the BP. PDMS, which is naturally hydrophobic,becomes hydrophilic after exposure to oxygen plasma for the purpose ofactivating the surfaces to enable bonding. The hydrophobicity recovers[36] after ˜24 hours, to reach a constant, time-independent contactangle of 107°. Based on this value, the computed BPs for CBVs #1, #2 and#3 are 713.4 (BP #1), 881.7 (BP #2) and 3035.7 Pa (BP #3), respectively.Experimentally measured values are somewhat lower than these estimates,mainly due to imperfections in the fabrication and, in particular,diverging angles that are slightly smaller than the design values, asshown in the SEM images in FIG. 25a [34]. For example, in CBV #2 and #3,the sharp edges where the straight channel and the diverging sectionintersect are rounded, with radii of curvature of approximately 35 μmand 27 μm, respectively (FIG. 31). Decreasing the channel opening angletends to decrease the bursting pressure. Therefore, the burstingpressure of the valve with a round edge is lower than that with a sharpedge (FIG. 31.)

Liquid that initially arrives at CBVs #1 and #2 encounters them in theirclosed states (FIG. 25e (i)). Upon reaching or exceeding BP #1, CBV #1opens to allow flow into the chamber (ii). After filling this chamber,the liquid flow bursts CBV #2 at sufficient pressure (BP of CBV #2 islower than that of CBV #3) (iii). By this process, all 12 chambers fillin a sequential manner, for flows that involve pressures larger than BP#2. For constant flow rate, this effect translates to time-sequencedsampling, or chrono-sampling. After use, the device can be removed fromthe skin and then inserted into a centrifuge (5000 rpm) to open CBV #3,thereby for moving liquid from each of the storage chambers intocorresponding extraction chambers to facilitate recovery for labanalysis (iv). The designs of the CBVs ensure that pressures generatedby the sweat glands exceed BP #1 and BP #2, but not BP #3, and thatcentrifugal pressures exceed BP #3.

Fluidic operation and stability under mechanical perturbation: FIG. 26ashows that these epidermal microfluidic chrono-sampling devices cansequentially collect liquid without undesired bursting of CPVs. The flowproperties of the microchannels ensure absence of unwanted mixing.Specifically, for channel dimensions of hundreds of micrometers and flowrates of 1-20 μl/min/glands, the flows are laminar, i.e. low Reynoldsnumbers (<1), and mixing occurs only by molecular diffusion [37, 38].The example here illustrates operation with water dyed using differentcolors and introduced in a time sequenced manner. Over relevant timescales (˜1 h) and temperatures (˜22° C.), diffusion occurs only within˜1 mm of the interfaces between water with different colors,corresponding to less than 10% of the total volume of the collectionchamber. The fourth chamber in this example is relatively dark due tomixing of the red dye in the bridge channel with green dye. Here, thered dye comes from the inlet and green dye follows the red, in sequence.After filling 11 chambers, removing the device and performingcentrifugation, each separate sample of sweat moves into a correspondingextraction chamber. FIG. 26c highlights the soft nature of these devicesand their ability to stretch, flex and twist without damage. Thefunction of the microfluidic channels, CPVs and chambers is unaffectedby these deformations or by shaking movements of the arm. Even theprocess of removing the device from the skin for chemical analysis,which includes significant mechanical stresses, does not affect thestability or fluidic containment (FIGS. 26c and 26d ).

Various applications of epidermal microfluidic sweat chrono-samplingdevice: These device platforms offer significant design versatility interms of overall dimensions, sizes of microfluidic channels andchambers, and numbers of chambers, as illustrated in FIG. 27 a. Thesmallest device shown here has three chambers, and a circular layoutwith a diameter of 1 cm, for use in space constrained regions such asbehind the ear. Due to the thin, soft mechanical construction, thisdevice and others with different designs are mountable on variouslocations on the body with equal fidelity, including the chest, back,forearm and thigh (FIG. 27b ). Additional features can be included. Forexample, in a standard layout, the adhesive covers the skin everywhereexcept for regions for collecting sweat. Possible effects related toblockage of sweat glands are reducible with the addition of channels ofrelief on the bottom surface of the device to define skin-interfacedmicrofluidic channels for transport of sweat to the outer perimeter(FIGS. 27c and 32 a.) Another option involves the introduction of openarchitectures via removal of regions of the device where the skinadhesive interface is not necessary (FIG. 32b ). Such layouts improvenot only management of flows of sweat in these locations but they alsoincrease the mechanical deformability. For a large device, it ispossible to locate an additional set of sampling capabilities in thecenter region (FIG. 27d ). The volume of the chamber can also be definedto meet requirements, with examples of 2.3 μl and 6.1 μl in FIG. 32 c.

The microscale dimensions of the devices, the water permeability of PDMSand the presence of microchannels and chambers with open outletscollectively lead to non-negligible rates of evaporation of sweat duringand after use. Experiments show that the evaporation rate from a typicaldevice is ˜3.5 μl/hr in a fully filled state, which corresponds to aper-chamber rate of ˜0.25 μl/h at 35° C., 40% relative humidity (FIG.33a ). This rate corresponds to ˜10% of the chamber volume per hour.Systematic studies show that most evaporation occurs through the sampleoutlets (FIG. 33c ). For long-term storage, mechanical clamps designedto block these regions (FIGS. 31c-e ) reduce the rate to 0.55 μl/hr at25° C., 30% relative humidity such that more than 50% of the samples canbe retained in the device for 1 day. Storage in high humidity conditions(99%) is possible for several days (FIG. 27d ).

In situ chrono-sampling from the skin and chemical analysis of capturedsweat samples: Human testing involved evaluations on volunteer subjectsduring running exercises and sessions in a sauna room. A formulation ofCoCl₂ in polyhydroxyethylmethacrylate (pHEMA) coats the center regionsof the chambers to produce a color change upon contact with sweat,thereby facilitating visualization of the filling process. For arepresentative running test, the first chamber filled after 8 min 20 sand the last chamber filled 10 min later. In a sauna, the first chamberfilled after 13 min 33 s and the last chamber filled after 8 min 30 s.in both cases, the sweat rate increases with time and the time forfilling each chamber after the first is less than one minute. In openingarea (2 mm in diameter) of the adhesive, there are approximately 5 sweatglands.[39] In the running exercise, the average sweat rate over thefirst six minutes is 0.50 μl/min/gland and increases to 0.63μl/min/gland. In the sauna, corresponding values are 0.54 μl/min/glandand 0.88 μl/min/gland. The initiation of perspiration is delayed in thesauna. Also, the initial sweat rates are similar in both conditions.But, the increase of sweat rate with time is high in the sauna. Thesevalues are tens of times higher than the rates from studies withconventional techniques (FIG. 34 top panel).[40] The increase of thesweat rate in the device is perhaps from the side effect of blockedregion by adhesive film.[41] We find that there is more sweat in theskin around the area blocked by adhesive tape (FIG. 34 bottom panel). Inthe device, there is 3 mm² opening hole for sweat enters to themicrofluidic channel and the other region about 700 mm² is covered byadhesive. To remove the enhance sweat collecting rate in the device, theadhesive region around the collecting region should be reduced. For thatwe design a different design to guide the sweat from the out ofcollecting region (FIGS. 27c and 32a ). Using the device, the sweat ratefrom the chrono-sampling device is similar with the result fromconventional method.

Centrifugation and extraction of samples from the extraction chambers ofdevices without CoCl₂/pHEMA allows mass spectrometer analysis of theconcentrations of key biomarkers. This centrifuge approach does notallow analysis of glucose, urea, calcium and magnesium becausemicroliter sample volumes with analytes at physiologically relevantranges of concentration fall below limits of detection. For devicesmounted on the forearm, samples collected after a running exerciseindicate a systematic decrease in lactate concentration for chambers1-9, followed by a slight increase for chambers 10-12. The concentrationof sodium remains constant until chamber 6, decreases for chamber 7 andthen increases again throughout. The decrease in concentration oflactate is consistent with the known dilution effect with increasedsweating.[19] The overall concentration of lactate is higher for sweatgenerated by sauna exposure compared to running, and the reverse trendapplies for sodium; both observations are consistent with previousstudies using primitive sweat harvesting techniques.[6] Theapplicability of the epidermal microfluidic systems to locations acrossthe body allows, as an example, comparisons of sweat from forearm,thigh, back and chest. Sodium and lacteal is more concentrated in thesweat collected from the chest and forearm, respectively, this agreeswith data from other researchers.[39, 42, 43]. The data reveal nosubstantial differences in potassium concentration for these bodypositions. Interestingly, in all cases, peaks in concentration oftenoccur at chambers 2 or 3, corresponding to the initial stages ofsweating. This result might be explained by differences in biomarkerconcentrations between sweat stored in the glands and newly generatedsweat. Alternatively, these changes indicate underlying physiologicalvariations. Previous technologies, due to their lack in volumetricand/or temporal precision, do not allow observation of subtle effectssuch as these. Even the most sophisticated wearable electronic systemsfor sweat analysis do not separate the sweat with time, such that sweatreleased at different times mixes together at the point of measurement[10-16].

FIG. 29 provides in situ perspiration analysis from various bodypositions during running exercise and thermal exposure in a sauna.Chrono-sampling of sweat generated on the forearm during a runningexercise under constant load (a) in thermal exposure at 56° C. (b).Cobalt (II) chloride dissolved in pHEMA and loaded into the devices aidsin visualization of the filling process. Chemical analysis of lactate,sodium and potassium in the sweat extracted from a chrono-samplingdevice mounted on the forearm during a running exercise (c), fromforearm (d), thigh (e), back (f) and chest (g) in thermal exposure. h)Regional variations of biomarker concentrations in sweat (lactate,sodium and potassium) collected from different body positions; chest,thigh, forearm and back.

In summary, this example introduces a soft, thin, skin-compatible, or‘epidermal’, microfluidic device platform for time sequenced capture,storage and retrieval of microliter volumes of sweat. A key advance isin the development and use of microfluidic capillary bursting valvestailored to operate in a range of pressures commensurate with thosenaturally generated by sweat glands in human skin. Trials on volunteersubjects in various scenarios and regions of the body reveal temporaland spatial changes in the concentration of key biomarkers. The resultsdemonstrate interesting time-dependent processes of relevance toexercise physiology and health/wellness more generally. Combined use ofthese platforms with colorimetric schemes for chemical sensing and withepidermal electronic components provide a broad range of engineeringoptions in the design of advanced systems of use for personal andclinical use cases.

EXAMPLE 3 Practical Strategies for Robust Colorimetric Detection ofSweat Biomarkers in Time Sequence Using Skin-Compatible MicrofluidicDevice

A microfluidics system is designed as suitable for quantitativecolorimetric detection of chloride in sweat which is one of sweatbiomarkers can predict dehydration when in the vigorous exercise andthermal condition. For the accurate detection of chloride, chlorideassay solution based on the competition reaction of TPTZ chelation wasmodified to be used for raw sweat in situ. And two valves for thequantitative analysis and chrono-sampling are introduced in themicrofluidic system. One is selective SAP valve which can close thechannel when the flow of sweat is reached to the point of valve, and theother one is hydrophobic valve at which the sweat flow in microfluidicchannel can select its direction by the hydrophobic resistance. Thequantitative analysis using the device showed change of biomarkers intime course in human study, and should be utilized at the healthcarestudies.

When the TPTZ has chelation with ferrous ion, the solution shows bluishcolor which is between 400-450 nm wavelengths. And the intensity ofcolor development is concerned with the quantity of TPTZ chelation onthe ferrous ion. Chelation is a kind of affinity competition, and ifthere is other ion substance which has strong affinity compared tochelating ion, TPTZ should migrate to the preferred ion. Mercury ion ismore preferred than ferrous ion, and TPTZ would make the chelation withmercury ion when both mercury and ferrous ions are in the same solution.The solution shows no color and would be remained as transparentsolution. It means almost TPTZ has chelation with mercury ion. Chloridehas strong ion interaction with mercury which is stronger than thechelation of TPTZ. If chloride ion is added into the solution containingTPTZ, ferrous ion, and mercury ion, then, TPTZ chelation with mercuryion would be migrated to the ferrous ion, and bluish color intensitywould be increased as the chloride concentration increases in thesolution system. In the case of the raw sweat assay, the competitionreaction may not be as effective as increase of all reactantconcentration. Then, the competition reaction of TPTZ chelation shouldbe re-designed as it is acceptable for the corresponding concentrationof chloride when the raw sweat is directly used.

The microfluidic system is expected to have good performance atcapturing and storing of sweat as soon as excreted from sweat gland.Conservation of sweat properties is very critical for sweat researchbecause sweat is easily evaporated and contaminated as its own function.Thus, the strategies that capturing the sweat as soon as perspirationfrom sweat gland in epidermis have potential value. In the conventionalstudy of sweat, patch type with either cotton or sponge to collect sweathave been used, and some kinds of electrochemical patches developedcurrently also limited in the patch type device which must be followedthe chance of exposure to atmosphere which can induce evaporation, andcontamination. The microfluidic system is not exposed to outsidecondition, and can be stored for relatively long time after sampling. Interms of storage with property maintaining, microfluidic system onepidermis is excellent. But there may be several drawbacks for precisionanalysis of sweat in situ if the colorimetric analysis paper is on themicrofluidic channel in which sweat flows. The colorimetric analysisbased chemical and enzymatic reaction is very sensitive for the volumeor mass ratio of the reactant involved in the color expression reaction.Continuous flow of sweat in the channel can be an inappropriatecondition for stable color development which is maintaining of thedeveloped color. As the flow of sweat is ongoing, the colorimetricmaterials in the filter paper matrix should be washed out and developedcolors would be diluted. It means the quantities of reactants containingbiomarkers in sweat need to be controlled and conserved to be shown assame color index after color development. Then, microfluidic channelshould be designed to support accurate color development and colormaintaining.

Also, sweat composition is continuously changed as the subject gets inthe situation of exercise or thermal condition. Then, chrono-samplingconcept would be needed to see the change of sweat contents in timesequence. In nature, the manner of channel flow would be divided when itmeets branched channel, and the dividing would be continued whenever itmeets branched channel if the channel has several reservoirs whichbranched from the main stream channel. Finally, the information of sweatin flow may be mixed with former and later flow, and be interfered.Introduction of microfluidics may helpful for the concept of sampling intime sequence.

For the precision analysis of biomarkers of sweat in time sequence,various strategies were integrated in the microfluidic device. Thecompetition reaction of TPTZ chelation for chloride assay was designedfor raw sweat analysis considering the affinities between Fe²⁺ and Hg²⁺.The instabilities of color development as increase of totalconcentration of ions also treated with the effect of surfactant andother ions. L-lactate assay cocktail was also modified in the ratio ofenzymes and dyes for raw sweat analysis. Furthermore, several mechanicalfunctions containing two key valves in microfluidics which were devisedand integrated to maximize the effect of microfluidic system on thequantitative analysis of raw sweat. Poly sodium acrylate which is a kindof super absorbent polymer (SAP) was introduced for selective valvewhich can vent the air and block the leakage of sweat from the channelin same time. Also direction selecting valve usinghydrophobic/hydrophilic characteristics was used for time sequentialsampling of sweat to see the difference of biomarkers in time course.Finally, human tests were conducted with instrumental analysis tocalibrate the quantitative analysis of biomarkers.

Skin-compatible microfluidic device for biomarker assay in time course:The device is comprised with several functional parts which areengineered using chemical and mechanical ideas to achieve accuratebiomarker assay in perspiration. It has five different time points forsweat chrono-sampling and each of the time points has three reservoirseither to store collected sweat samples or to analyze biomarkers incolorimetric assay. FIG. 35 shows the overall view of microfluidicdevice design. The microfluidic channel system is structured in a doublelayer as the function of SAP valve which is selective valve to block thevent when sweat flow is reached at the valve point (FIG. 35a ). The PDMSmolds which are cured by soft-lithography are aligned and stacked up asflipping over as the channel side faces down, and bottom layer of ˜200μm thickness covers the channels of SAP layer. Also there is a piece offilm which character is flexible but not stretchable on the SAP valvepoint of the bottom side to support and concentrate the swelling forcetoward upside of device. The inlet of microfluidic channel which isbottom side of the device should be anchored on the epidermis stronglyand tightly because the device is designed as passive system which canbe driven by the pressure of pumping up from sweat gland, and once thesweat is pumped from sweat gland, all system can be operated toward oneorientation of inlet to outlet with filling up the reservoirs locatedalong the main stream flow in series. Then, the anchorage of inlet onthe sweat gland is very important to operate this device, and specialadhesive for medical use is introduced. The fabricated device can bestretched, and is compatible with the skin shape and movement. Theskin-compatible property make the device is available in the exerciseenvironment in which vigorous movement should be followed. Also designedchemical and mechanical factors supported the accurate biomarker assayin situ.

FIG. 35b is the illustration of assembled device. Dimension of thedevice is 30 mm of diameter, 100-200 μm of channel width, ˜300 μm ofchannel depth, 2.4 mm of single reservoir diameter, and ˜500 μm of totaldevice thickness. The microfluidic system can store total ˜40 μL ofsweat and ˜6 μL can be stored for one time point. Once the reservoirsare filled up with sweat, inlet and outlet would be closed to preventcontamination and mixing by SAP valve and all reservoirs in each timepoints (T1-T5) would be filled up in the chrono-sampling manner. Onetime point station is consisted with hydrophobic valve, three reservoirsto analyze sweat, and three SAP valves for each reservoir (FIG. 35c ).The reservoirs are used for colorimetric assay chamber, and it can bedesigned to use as sweat storage chamber without colorimetric assaypaper. The device was also designed as chrono-sampling manner. The redarrows in the FIG. 35c indicate the flow order in time sequence. Thesweat flow comes to the inlet of T1 point, it goes to the reservoirs atfirst, and never goes to next time point by hydrophobic valve unless T1point is not filled up with sweat. In the process of filling up, thethree colorimetric assay papers get wet and present the condition ofcorresponding quantities of biomarkers in sweat at that time moment.This flow manner would be repeated in the series of reservoirs on thedevice.

Chemical design for accurate and stable color development, and imageprocessing: Human sweat contains around 50-100 mM concentration ofchloride as the subjects are in the environment of exercise or thermalcondition. Usually TPTZ method is conducted around 1 mM chlorideconcentration, and the color development could be observed at the rangeof chloride concentration of the sample. And the development of colorcould be calculated as color intensity or converted to the absorbancemeasuring from spectrophotometer. To assay high concentration chloride,total concentration of assay solution based on TPTZ method should beincreased as corresponding quantity of chloride in the sample of rawsweat. But high concentration of complex solution containing variousions showed some issues about interference and unstable colordevelopment because of physical space saturation for the interactions ofion molecules in the limited solution volume. The complex of TPTZchelation molecule must shows dipolar behavior and the distance of thecomplex which emit bluish color would be reduced in probability, and asclosed the mercury ion and TPTZ chelated ferrous ion, TPTZ chelationwould get destabilized, and finally the bluish color would be lost.Then, once the color has developed, the color index was easily changedas time. The TPTZ competition system might be affected as the increaseof the competition ions. Though TPTZ is liberated from mercury aschloride is added, high concentration of mercury could still affect thechelation of TPTZ with ferrous ion and the changed solution color couldnot be maintained, and get dilute as solution stabilized. Then colordevelopment gets destabilized as total solution gets stabilized whenhigh concentration of assay solution is used. Then, some stabilizationagent was introduced. Other ions which do not affect the TPTZ chelationshould be contained to buffer the effect of competition ions and shouldact as it secures physical space of molecules in the limited solution.

Also there are some issues of TPTZ solubility in water based solution.TPTZ is insoluble in water, and stock solution is usually prepared addedinto methanol. Though pure TPTZ has insoluble in water, once it haschelation ligand, it seemed allows the solubility of ligand substance.But the chelation complex may show such a dipole effect, then, thecomplex has solubility and insolubility in the water in same time. Thensurfactant should be effective to stabilize the difference of TPTZsolubility. Also the stabilization of TPTZ using surfactant induces thestabilization of color development. FIG. 36a shows the effect of tween80 concentration on the stability of color development. Variousconcentration of tween 80 was tested. When the assay with raw sweat anddesigned ratio of assay solution was conducted without the surfactant,the color development was not maintained, and the color index of assaypaper was gradually decreased as time course. But when the tween 80 wasused with the same condition of chloride detection, the developed colorshowed fairly maintained at over 0.8% tween 80. And When 1% of tween 80was used, the color was maintained by 24 h (there is by 3 h in FIG. 36a).

Color index of RGB value is changeable as environment of the lightsource for image capturing. FIG. 36c show the difference of RGV valuewhen the same chloride calibration color is exposed to the differentlight color of white and yellow. The difference of the color index needsto be calibrated as the interference of various light sources. Then, CIEcolor calibration method was employed, which can correct the image fromvarious light sources using white value standard. CIE L*a*b* color spaceis a three dimensional real number space that can incorporate both RGBand CMYK systems. Then, the complicating difference of color index couldbe described as converting into single index using Eq. XI. FIG. 36d isthe comparison of color index conversion from the color image from whiteand yellow light source. The difference of color information under whiteand yellow light was converted as defined index, and it showed almostsame values for the various levels of color development. FIG. 36e is thecalibration curve for chloride assay in the raw sweat.

Mechanical strategies for accurate and stable color development: Colordevelopment which can be converted into as RGB index as theconcentration of desired biomarker is based on the stoichiometry ofchemical and enzymatic reactions. Then, the different quantities andratios of the reactants can be shown as the particular color expressionas the results of the reactions, and the control of reactants ratio, andquantity is the key point for the accurate color development. Wellcontrolled assay solution containing the reactants was dropped on thefilter paper, and accurate sweat amount must be sampled to be reactedwith the reactants permeated in the filter paper. But the colordevelopment might be diluted and would show inexact information if theassay paper is on the continuous flow because the reactant in the assaypaper might be washed out by the sweat flow. Then, the continuous flowshould be stopped when the reservoir has filled up with sweat sample.Also the sampled sweat should be separated from main flow in same timeto avoid mixing of sweat information between former and later sweatflows. Each reservoir has two pathway of inlet for sweat coming in frommain flow of microfluidic device and outlet as air vent. And bothpathway should be closed by selective valve to secure allocate amount ofsweat sample. To realize the selective valve which can pass the air inchannel but stop the flow, the microfluidic channel should be collapsedphysically such as pneumatic valve. In the pneumatic valve operation,there are flexible thin layer between flow channel and particular airroom at the valve point, and the channel side would be collapsed whenthe air pressure is increased as blowing air in the air room. The keymechanism of pneumatic valve is expansion of air room, and the selectiveSAP valve borrows the concept of some space expansion which can inducethe physical collapse to close the valve logically.

SAP materials have the character of swelling up in gelation process whenit meets the water based solution which is equal with the effect ofspace expansion. Furthermore, SAP is a powder in normal condition, andit can be used as vent though it filled a part of channel. Then,introduction of SAP can achieve not only the effect of a selective valveto block the sweat flow, but also the effect of pneumatic valve to closeinlet point. FIG. 37a shows the design of selective SAP valve and FIG.37b is that the selective SAP valve is activated. The valve is designedsuch a pneumatic valve which have collapse area and expanding area. Andthe expanding area is filled with SAP. It is beneficial to be bi-layerstructure, and SAP layer is responsible for the

Once the sweat fills the reservoirs up, both inlet from main streamchannel and outlet for ventilation of air would be closed as the effectof SAP swelling. The reservoirs for one time point have double layerstructure to realize the SAP valve. The sweat flow would come in fromupper layer channel, and if the reservoir is filled up or the assaypaper is wet by the pressure of incoming flow, the SAP would beactivated to be swollen by absorption of sweat flow. The activation ofSAP valve would finally induce the closing of ventilation channel, andthe inlet channel also would be collapsed to be closed (FIG. 37b ). Twoactions of closing ventilation and collapse of inlet would not beoccurred in same time, but as the SAP is gelated, it would be swollenand the collapse would take place in short time.

Sweat sampling in time sequence: As the continuous change of sweatcomposition over time, chrono-sampling concept should be integrated.FIG. 38a is the concept of chrono-sampling that if there is branchedmicro channel, and flow would select its preferred property such ashydrophobic or hydrophilic characteristics of channel surface. In caseof sweat, it is biofluid based on water, and it would select relativelyhydrophilic channel when the flow meets the branched channel in whichone is treated as hydrophilic, and the other one is hydrophobic. Oncethe flow select hydrophilic channel at the point of branch, it never goto the hydrophobic channel (FIG. 38b ). But the end of hydrophilicchannel is blocked, the inside pressure of channel gets increased, andthe pressure of flow would burst out though the hydrophobic channel atlast. The selection of flow direction could be expressed as resistance,and at first, flow selected channel of less resistance, and as theresistance is increased for the blocking, the flow pressure overcomesthe hydrophobic channel resistance (FIG. 38c ). This simple conceptshould be utilized and employed to realize the flow direction selectivevalve, named hydrophobic valve in this study. The backbone structure ofskin-compatible microfluidic device is PDMS, and OH group which acts ashydrophilic functional group is developed when it is exposed to theoxygen plasma. A procedure of PDMS surface modification is available.The original character of PDMS is hydrophobic, and the regions forhydrophobic valve should be masked conformally using PDMS andanti-adhesion agent. After expose to the plasma and relief of themasking, the masking region would be remained as the originalhydrophobic region, and other part would be modified as hydrophilic.FIG. 38d shows the illustration of hydrophobic valve operation steps intime sequence. Each time point of the microfluidic device has same flowchannel shape, and the operation steps would be repeated as the timepoints are filled up. When the flow comes to the first time point (T1),hydrophobic valve would stop the flow and the sweat flow would bedivided to three branched channel and go through the SAP valve points.If the reservoirs are filled up with the sweat sample, and selective SAPvalve is activated, the total resistance of T1 for the sweat flow wouldbe increased and the sweat pumping pressure would overcome theresistance of hydrophobic valve. Then, the main flow will jump to nexttime point. As the effect of hydrophobic valve and selective SAP valve,finally the reservoirs at all points (T1-T5) would be filled up as thechrono-sampling manner (FIG. 38e ). FIG. 38f is the results offabricated microfluidic device which have the concepts of hydrophobicvalve and selective SAP valve. Blue color dyed artificial sweat waspumped into the device. The pressure of artificial sweat pumping was˜300 Pa which is derived from potential energy of 3 cm heights and theresults showed the chrono-sampling manner of sweat flow as we designed.

FIG. 38g is the effect of pumping pressure which drives the microfluidicdevice. The device is designed as passive valve system, and the fillingup time for five time points depend on the pumping pressure at inlet ofmicrofluidic channel. The pumping pressure from sweat gland is relatedwith the sweat excretion rate, and the correlation with the pressure andsweat excretion rate would be proportional (ref.). Then, the pressure isrelatively high, the flow rate would be rapid, and filling up time wouldbe shortened. On the other hand, at the low pressure, the filling uptime would be extended. Also the device may be modified to be extendedby increasing channel size between time points as buffer area which isas the need of sweat research. Also limited range of the pumpingpressure could be controlled by changing the inlet size of themicrofluidic device on the bottom cover layer. The FIG. 38h is theeffect of inlet size on the sweat capturing time. The inlet size testwas performed on the real skin. Subjects experienced the thermalcondition of over 40° C. temperature of thermal condition.

Preparation of chemical and enzymatic assay agent, and devicefabrication: Polysodiumacrylate was determined as SAP material andsynthesized as the method of Kabiri et al. (2003). Glacial acrylic acidmonomer (Sigma Aldrich) was neutralized with 55% KOH, and 2 mL Ammoniumsulfate (37.5 g/L) and 4 g sodium bicarbonate were added into theacrylic acid solution. N, N′-methylenebisacrylamide, fast swelling agent(Sigma Aldrich) of 2 mL Sodium metabisulfite (31.5 g/L), cross linker of2 mL were added, and viscosity was increased for the gelation. Afterthat, the gel was spread over the oven tray, and dried at 70° C. for 24h. Dried material was ground and meshed with No. 000 sieve (000 μm meshsize).

Chloride assay agent was prepared based on the method of 0000 et al.TPTZ was resolved in methanol to be 0000 M concentration. HgSO₄ andFeSO₄ solution was prepared in 10 mM concentration. [More Chloride assaymethod]

Filter paper (Whatman No. 1) was used as assay agent matrix afterpunching in 2.5 mm diameter. Exact amounts of 2.6 μL solution forchloride assay and 2 μL for lactate assay were dropped on the filterpaper of 2.5 mm diameter and dried at the ambient room temperature for 1h. In the case of l-lactate, additional enzyme cocktail of HRP (20mg/mL) and LOx (60 mg/mL) are dropped in 1:2 v/v ratio. Also pH assaypaper was purchased, and punched to be 2.5 mm diameter to insert thereservoir in microfluidic channel.

Fabrication of skin-compatible microfluidic device: To generate soft andflexible backbone for skin-compatible microfluidic device, softlithography of replica molding process was conducted which make mold ofparticular channel shape of polydimethylsiloxane (PDMS; Sylgard 184, DowCorning; mixed at a 23:1 ratio of base to curing agent by weight) frommold cast of silicon wafer. For that, photolithography was performed.Cleaned silicon wafer was coated with KMPR 1010 (MicroChem, Westborough,Mass.) which is negative photoresist, and the coated wafer was exposedto the UV with channel designed mask for 30 sec. Developed wafer wasetched using deep reactive-ion etching (STS Pegasus ICP-DRIE, SPTSTechnologies Ltd, UK) to be 300 μm depth microfluidic channel. Afteretching, the mold cast was coated with polymethylmethacrylate (PMMA) at3000 rpm spin coater for 30 sec, and curing at 180 for min was followed.The conditions of casting on spin coater were that 250 rpm for channellayer, and 400 rpm for SAP layer. After 30 sec spin coating at eachcondition, curing was followed at 70° C. for 4 h. Masking of hydrophobicvalve spot for each time point on channel layer was conducted using PDMSafter anti-adhesion treatment. Prepared SAP material was loaded at SAPvalve point on the SAP layer, and SAP layer and bottom cover was bondedafter oxygen plasma treatment. Channel layer was also treated withoxygen plasma, and masking material was removed. Bonding of channellayer with SAP layer assembly was followed after loading of colorimetricdye and color reference. Adhesive layer was assembled on bottom coverlayer, and the assembly was punched as 30 mm diameter.

Data process, image process and instrumental analysis: After subjecttest and pumping test, the photo of device was taken, and the black andwhite balance for the image was performed following the RGV values ofcolorimetric detection for all biomarkers were obtained using AdobePhotoshop program. The values were then converted to CIE L*a*b* values,and color difference was calculated using following equation

ΔE* _(ab)=√{square root over ((L* _(n) −L* ₀)²+(a* _(n) −a* ₀)²+(b* _(n)−b* ₀)²)}  (XI)

Where L* is lightness, a* is green to red scale from negative topositive values, and b* is blue to yellow. L*n, a*n, and b*n denotes thedeveloped assay's values at level n, and L*0, a*0, and b*0 are thevalues for a white reference paper.

Human study: On body test for human study for was performed with AirForce Research Laboratory (Write-Patterson Air Force Base, Dayton,Ohio). The subject experienced the environment of marching condition aswearing full battle gear including a helmet, Kevlar vest, 50 lb. pack,and M4 rifle on uniform. The ruck march was conducted on a treadmillprotocol with two inclines and speeds of easy and moderate. Beforedowning the gear, Wescors, sweat sampler and skin-compatiblemicrofluidic device were placed on the right forearm, and they arecovered with a compression arm sleeve. The march was maintained untilthe subject was exhausted with dehydration, and it was approximately1.5-2 h march.

Exemplary devices: Referring to FIGS. 1 (panels A-D), 2 (panel D), and 3(panel A), device 10 may be affixed to a surface or epidermal layer 45of skin 40 to handle a biofluid, indicated by arrow 20 representingbiofluid released from the skin, including sweat. Biofluid 20 mayinclude one or more analytes and/or one or more biomarkers, such aslactate, pH, or chloride. Device 10 may include a functional substrate30, which may be formed of a polymer, including PDMS, and may support amicrofluidic network 150, biofluid collection structures 60, and one ormore sensors 50. FIG. 2 panel D shows microfluidic network 150,supported by functional substrate 30, with microchannels 160 in fluidcommunication with biofluid collection structures 60, including at leastone microfluidic channel 62 and a reservoir, illustrated as fourreservoirs or chambers 64. Biofluid collection structures 60 may form atleast a part of microfluidic network 150. Biofluid collection structures60 and sensor(s) 50 may be positioned on a support surface 35 offunctional substrate 30. One or more inlets 130 may be used to convey,transport or exchange biofluid 20 to sensor 50 released from skinsurface 45, with inlets aligned in each of the relevant layers. Anadhesive layer 140, having openings or harvesting areas 145 positionedto fluidically align or correspond with inlet(s) 130. Microfluidicnetwork 150 may further include outlets 260, such openings and/ormembranes. Device 10 may further include a cover or capping layer 190,which may be formed of a polymer, including PDMS.

FIG. 1 panel A further illustrates control and/or communication meansfor interfacing or interacting with, including broadly speaking,actuating, device 10, including as desired, for example, an externaldevice such as a smartphone or tablet computer. Interfacing unit mayinclude a coil 340, an actuator 292 (for either controlling electroniccircuitry and/or a physical parameter exerted on a skin surface, alsodescribed herein as an NFC chip), a transmitter, a receiver, and/or atransceiver. Interfacing unit may provide NFC capability, such as byincluding in NFC electronics, such as an NFC chip and a near-fieldcommunication coil 340.

Sensor 50 may include a plurality of sensors, such as a first sensor 70(FIG. 3, panel A, top left) and second sensor 80 (FIG. 3, panel A, topright). Sensors 70 and 80 may be used for determining concentration ofone or more different analyte, or analyte concentration over differentconcentration ranges. Different sensors may be used to measure differentbiofluid properties. Sensors 50 may correspond to one or morecolorimetric sensors 280. Colorimetric sensors 280 may include one ormore indicator reagents 284, including color-responsive reagents 282,such as a colorimetric dye. Color-responsive reagent(s) 282 may beprovided in a biofluid collection structure 60, such as microfluidicchannel 62 or reservoir 64. A hydrogel 68 may be provided within abiofluid collection structure 60 to immobilize color-responsive reagent282.

FIGS. 24 and 25 illustrate a microfluidic network 150 havingmicrochannels 160, reservoirs 200, and valves 210. Each of valves 210 isa passive valve or an active valve, and may be a capillary burst valve.FIG. 25 illustrates examples of various valves 210. The valves mayinclude direction selective valves. FIG. 37, panels A and B, illustratesa valve that is a super absorbant polymer (SAP) valve. FIG. 38, panelsA, B, and D, illustrates a valve that is a hydrophobic valve 218.

FIG. 40 illustrates a microfluidic network 150 including a first layer170 embossed with relief geometry 180, comprising relief features 310,to define microchannels 160. A second top capping layer 190 may be usedto enclose the microchannels 160.

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. When a compound is described herein suchthat a particular isomer, enantiomer or diastereomer of the compound isnot specified, for example, in a formula or in a chemical name, thatdescription is intended to include each isomers and enantiomer of thecompound described individual or in any combination. Additionally,unless otherwise specified, all isotopic variants of compounds disclosedherein are intended to be encompassed by the disclosure. For example, itwill be understood that any one or more hydrogens in a moleculedisclosed can be replaced with deuterium or tritium. Isotopic variantsof a molecule are generally useful as standards in assays for themolecule and in chemical and biological research related to the moleculeor its use. Methods for making such isotopic variants are known in theart. Specific names of compounds are intended to be exemplary, as it isknown that one of ordinary skill in the art can name the same compoundsdifferently.

The following references relate generally to fabrication methods,structures and systems for making electronic devices, and are herebyincorporated by reference to the extent not inconsistent with thedisclosure in this application.

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2014 — —

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anydevice components, combinations, materials and/or compositions of thegroup members, are disclosed separately. When a Markush group or othergrouping is used herein, all individual members of the group and allcombinations and subcombinations possible of the group are intended tobe individually included in the disclosure

Whenever a range is given in the specification, for example, a numberrange, a temperature range, a time range, or a composition orconcentration range, all intermediate ranges and subranges, as well asall individual values included in the ranges given are intended to beincluded in the disclosure. It will be understood that any subranges orindividual values in a range or subrange that are included in thedescription herein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when compositions ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements and/or limitation or limitations,which are not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

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1.-100. (canceled)
 101. A device for multiparametric and temporalcharacterization of sweat comprising: a soft and flexible functionalsubstrate for adhering and conforming to a surface of the skin, saidfunctional substrate comprising a microfluidic network channel having aplurality of microchannels and reservoirs configured for time-dependentcollection of said sweat; and a plurality of sensors supported by saidfunctional substrate and configured for multiparametric and temporalanalysis of said sweat; and wherein said microfluidic network channelprovides for microfluidic transport of at least a portion of said sweatto said sensors.
 102. The device of claim 101, wherein the sensorsdetect: a sweat rate or sweat volume; and a sweat composition.
 103. Thedevice of claim 102, wherein the sweat composition comprises aconcentration or amount of at least two biomarkers in said sweat. 104.The device of claim 103, wherein the biomarkers correspond toelectrolytes, metabolites, or both electrolytes and metabolites. 105.The device of claim 101, further comprising a plurality of microchannelopenings on a skin-facing surface of the functional substrate for sweataccess to the microfluidic network channel.
 106. The device of claim101, wherein at least one microchannel comprises colorimetric assayreagents to detect water, lactate, chloride, glucose, creatinine or pH.107. The device of claim 101, further comprising a near-fieldcommunication component for wireless communication between the deviceand an external device having an image capture and analysis software.108. The device of claim 101, wherein at least one microchannel isconfigured to receive sweat and amount of sweat is determined by fillingof the at least one microchannel over time.
 109. The device of claim108, further comprising a color-responsive reagent provided along thelength of the at least one microchannel to provide an opticallydetectable leading edge of sweat.
 110. The device of claim 108, whereinthe at least one microchannel has a serpentine configuration to maximizethe at least microchannel volume for receiving sweat without increasingthe functional substrate footprint.
 111. The device of claim 108,wherein the at least one microchannel has an effective volume forretaining a volume of sweat corresponding to an exercise time of up to 6hours.
 112. The device of claim 108, further comprising passive burstvalves connected to the microchannels and/or reservoirs to providecontrol of sweat flow direction microfluidic network channel.
 113. Thedevice of claim 101, wherein the reservoirs each contain acolor-response reagent for quantifying an amount of a biomarker in thesweat.
 114. The device of claim 113, wherein at least one microchannelcontains a color-response reagent for quantifying an amount of sweat inthe at least one microchannel and at least two reservoirs for detectingconcentration of at least two biomarkers.
 115. A method for analyzingsweat, the method comprising the steps of: providing the device of claim1; conformally contacting the functional substrate with a skin surface;collecting sweat released from the skin surface in the microfluidicnetwork; exposing the plurality of sensors to the collected sweat; andanalyzing the plurality of sweat biomarkers and sweat loss with theplurality of sensors; thereby analyzing sweat.
 116. The method of claim115, wherein the analyzing step comprises detecting a colorimetricchange in a colorimetric sensor.
 117. The method of claim 115, whereinthe analyzing step further comprises detecting a leading edge ofcollected sweat in a microchannel as a function of time.
 118. The methodof claim 116, wherein the analyzing step further comprises opticallydetecting the colorimetric change in a colorimetric sensor with anexternal device having image capture and analysis software.